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Journal of Virology, January 2000, p. 401-410, Vol. 74, No. 1
0022-538X/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The Differentiation-Specific Factor CDP/Cut Represses
Transcription and Replication of Human Papillomaviruses through a
Conserved Silencing Element
Mark J.
O'Connor,
Walter
Stünkel,
Choon-Heng
Koh,
Holger
Zimmermann, and
Hans-Ulrich
Bernard*
Institute of Molecular and Cell Biology,
Singapore 117 609, Singapore
Received 9 August 1999/Accepted 29 September 1999
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ABSTRACT |
The life cycles of human papillomaviruses (HPVs) are intimately
linked to the differentiation program of infected stratified epithelia,
with both viral gene expression and replication being maintained at low
levels in undifferentiated basal cells and increased upon host cell
differentiation. We recently identified, in HPV-16, a negative
regulatory element between the epithelial-cell-specific enhancer and
the E6 promoter that is capable of silencing E6 promoter activity, and
we termed this element a papillomavirus silencing motif (PSM) and the
unknown cellular factor that bound to it PSM binding protein (PSM-BP).
Here we show that the homologous genomic segments of six other
distantly related genital HPV types contain a PSM that binds PSM-BP and
is capable of repressing transcription. Conservation of the PSM
suggests that it is indispensable for the HPV life cycle. Purification,
electrophoretic mobility shift assay experiments, and the use of
specific antibodies proved that the cellular factor PSM-BP is identical
to a previously described transcriptional repressor, the CCAAT
displacement protein (CDP), also referred to as the human Cut protein
(Cut). CDP/Cut repression of HPV-16 may stem from the modification of
specifically positioned nucleosomes, as suggested by
transcriptional stimulation under the influence of the histone
deacetylase inhibitor trichostatin A. CDP/Cut is an important
developmental regulator in several different tissues. It was recently
shown that CDP/Cut is expressed in basal epithelial
cells but not in differentiated primary keratinocytes. This suggests
the possibility that repression by PSM couples HPV transcription
to the stratification of epithelia. In each of the studied HPV types,
the two CDP/Cut binding sites of PSM overlap with the
known or presumed binding sites of the replication initiator protein
E1. Transfection of CDP/Cut expression vectors into cells that support
HPV-16 or HPV-31 replication leads to the elimination of viral
episomes. Similarly, two PSM-like motifs overlapping the E1 binding
site of bovine papillomavirus type 1 bind CDP/Cut, and CDP/Cut
overexpression reduces the copy number of episomally replicating BPV-1
genomes in mouse fibroblasts. CDP/Cut appears to be a master regulator
of HPV transcription and replication during epithelial differentiation,
and PSMs are important cis-responsive targets of this repressor.
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INTRODUCTION |
Human papillomaviruses (HPVs) infect
mucosal and cutaneous epithelia and cause benign and malignant lesions,
such as common warts, genital warts, and cervical cancer (29,
67). Transcription of HPVs is regulated in a complex manner in
response to the identity of the infected epithelial cell type, the
differentiation state of the stratified epithelium, the physiological
state of the host, and the episomal or chromosomally integrated state
of the viral genome. Most of the cis-responsive elements
that mediate the activity of cellular and viral factors on HPV gene
regulation are located in the long control region (LCR), which lies
between the 3' terminus of the L1 capsid gene and the start of the E6
oncogene. With a size of 850 bp in HPV-16 and a similar length in other
genital HPVs, the LCR takes up about 11% of the 7.9-kb circular DNA
genome. When one compares remotely related HPV types, one finds that
their genes are clearly homologous but their LCRs have diverged so much that they cannot be unequivocally aligned and their nucleotide sequences lack long, similar segments. However, when one compares the
LCRs of more closely related HPV types, such as the large group of genital HPVs, one finds many short similar
cis-responsive elements, which are activated by a defined
subset of the cellular transcription factors of the infected cell. This
conservation of sequence and function suggests that these particular
mechanisms are so appropriate for the biology of these HPV types that
they could not undergo evolutionary changes (13, 44). Here
we study such an element that is conserved among genital HPVs, and for the first time we describe an element that represses both transcription and replication, most probably in response to the differentiation of
stratified epithelia.
The central part of the LCR of genital HPVs functions as an
epithelial-cell-specific transcriptional enhancer (14, 23, 50), and the 3' part contains the replication origin and the E6
promoter (12, 16, 19, 31, 44, 59). We have recently detected
a novel transcriptional silencer, which we called a papillomavirus silencing motif (PSM), in this 3' part of the LCR of HPV-16
(45). The PSM is physically separate from and functionally
independent of another repressor domain, 50 to 100 bp further in the 5'
direction, which consists of five binding sites for the transcription
factor YY1 (9, 38, 46). We have defined the PSM as a 25-bp
segment, containing two direct repeats of the sequence
5'-TAYAATAAT-3' spaced by 7 nucleotides consisting of the
sequence 5'-ACTAAA*C-3', where A* is position 1 of the
genomic map of HPV-16. Each of the two TAYAATAAT repeats
binds the same factor, which we called the PSM binding protein
(PSM-BP). Binding of a PSM-BP dimer to the two flanking repeats
occurred cooperatively and led to synergistic transcriptional repression.
In this study we obtained evidence that all genital HPV types and
bovine papillomavirus type 1 (BPV-1) contain very similar sequence
motifs, and we observed identical binding properties for PSM-BP in the
eight papillomavirus types that we examined. All of these motifs are
located in a similar position between the enhancer and the E6 promoter,
and they overlap with documented or presumed binding sites for the
replication initiator protein E1. We identified PSM-BP as the
previously described protein CCAAT displacement protein (CDP/Cut), and
we show that CDP/Cut represses the transcription and replication of
genital HPVs. CDP/Cut is a known repressor of numerous cellular genes
that are developmentally regulated in a variety of cell types (8,
34). CDP/Cut binds to and represses these cell-type-specific
promoters in developmental stages when these specific genes are not
required. Tissue-specific activation of these promoters results upon
terminal differentiation when CDP/Cut availability decreases and
promoter binding is lost. A similar phenomenon has been reported for
stratified epithelia (1). Consequently, it is likely that
CDP/Cut is a general repressor of transcription and replication of
genital HPVs and a key regulatory device in coupling these two
functions to the differentiation program of the infected stratified epithelium.
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MATERIALS AND METHODS |
Plasmid constructs.
All constructs used in functional assays
were based on the chloramphenicol acetyltransferase (CAT) reporter
plasmid pBLCAT3dH/N, a modified version of pBLCAT3 (51). The
derivative p80 (46) contains HPV-16 promoter sequences from
nucleotides 16 to 80 cloned into the BglII and
XhoI sites of pBLCAT3dH/N and contains a conserved Sp1 and
two E2 binding sites and a TATA box, typical for all genital HPVs
(61). The construct p80SV was created by cloning the
EcoRI fragment containing the simian virus 40 (SV40)
enhancer from the oligonucleotide vector (64) into
pBluescript SK(+) (Stratagene). This fragment was then excised with
HindIII and BamHI and cloned into p80.
Double-stranded DNA oligonucleotides containing HPV-16 sequences
(genomic position 7883-15), either wild type or mutated (m), and with
complementary BamHI and BglII ends, were cloned into the BamHI site of p80SV to give the constructs p80SV16
or p80SV16m respectively. Similar double-stranded oligonucleotides (see
Fig. 2) containing homologous sequences flanking the E1 binding sites
of other HPVs (HPV-2, HPV-11, HPV-18, HPV-31, HPV-33, and HPV-45) were
cloned using an identical strategy to generate the plasmids p80SV2,
p80SV11, p80SV18, p80SV31, p80SV33, and p80SV45. The structures of all
constructs were confirmed by determination of their nucleotide
sequence. The structures of pBR322-HPV-31 (22, 27), used in
replication, and of the CDP expression vector pMT2-CDP and the parental
vector pMT2 (42) have been published.
Cell culture and gene expression assays.
C33A, an HPV-free
cell line derived from a cervical carcinoma, was cultured in
Dulbecco's Modified Eagle's medium containing 10% fetal calf serum,
plated onto 10-cm culture dishes, and transfected at 50 to 70%
confluency with Lipofectin reagent (GIBCO-BRL). For each transfection,
30 µl of Lipofectin was mixed with 5 µg of DNA in 1 ml of medium
and left at room temperature for 15 min before being added to 9 ml of
medium. After 18 to 24 h, the medium containing Lipofectin was
replaced with 10 ml of Lipofectin-free medium, and the cells were
incubated for a further 24 h before harvesting.
Chloramphenicol acetyltransferase (CAT) assays were performed as
modified by our laboratory (11). CAT activities were
reported as picomoles of chloramphenicol acetylated per minute per
milligram of protein extract and were measured by quantification of
radioactive spots on thin-layer chromatograms. Each value represents
the average of three to six independent transfections with at least two
different DNA preparations.
The cell line Cho 4.15 (E1/E2) was a gift from M. Ustav and I. Ilves.
ID13, a mouse fibroblast line derived from c127 with
episomally
replicating copies of the BPV-1, has been maintained
in our laboratory
for more than 10 years and was originally obtained
from P. M. Howley (
32a).
For stimulation by trichostatin A (TSA), cells were electroporated with
vector DNA and incubated in growth medium for 24 h
before the
addition of TSA in ethanolic solution to a final concentration
of 100 µg/ml; luciferase activity was determined after another
24
h.
Electrophoretic mobility shift assay (EMSA).
A 50-ng portion
of annealed oligonucleotide was labelled with [32P]dATP
and [32P]dCTP nucleotides by using Klenow polymerase.
Approximately 250 pg of purified labelled probe with an activity of
approximately 20,000 cpm was used in a previously described standard
reaction (46). Samples were run at 200 V for 2 h on a
4% polyacrylamide gel containing 0.25× Tris-borate-EDTA buffer (TBE).
The gels were transferred onto blotting paper, dried for 1 h, and
exposed to autoradiographic film. For supershift experiments, 1 µl
(1.5 µg) of anti-CDP antiserum (42) or the same amount of
a preimmune serum was added to the reaction mixture, radiolabelled
probe was added, and the mixture was incubated on ice for 10 min.
Purification of proteins by column chromatography.
To enrich
the PSM-BP, we either prepared nuclear extract from HeLa cells
ourselves (17) or purchased it from the Computer Cell
Culture Center (Brussels, Belgium). During fractionation, binding of
proteins to an HPV-16 oligonucleotide with the two PSMs was monitored
in EMSAs. As a first step toward purification, 10 ml of nuclear extract
corresponding to a protein content of 150 mg was applied to a
heparin-Sepharose column (Pharmacia), previously equilibrated with 5 column 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). After loading of nuclear extract, the
column was washed with 2 volumes of buffer D and bound proteins were
eluted stepwise with increasing concentrations of KCl in buffer D. PSM-BP EMSA activity was observed in the 0.3 M fraction. This fraction
was dialyzed against buffer D and fractionated by ammonium sulfate
precipitation. Binding activity was found in the 20 to 40% ammonium
sulfate precipitate after overnight dialysis against buffer D. Then
200-µl volumes of active fractions were loaded onto a Sephacryl S400
column (Pharmacia) equilibrated with buffer D. The EMSA activity eluted
with a molecular mass of around 200 kDa by comparison with a
-amylase size marker.
In vivo replication assays.
To support the replication of
HPV genomes or chimeric HPV constructs, we used the cell line Cho4.15,
which expresses the BPV-1 E1 and E2 proteins from stably transfected
intrachromosomal E1 and E2 genes (48). The cells were
transfected by electroporation of 3 µg either of pBluescript KS
harboring a 500-bp XhoI-HindIII fragment of
the LCR of HPV-16 with the enhancer, the replication origin, and
the E6 promoter or of HPV-31 genomes free of plasmid sequences. The
latter were obtained by separating them from vector sequences of the
pBR322-HPV-31 construct by EcoRI digestion and religation of
the 8 kb viral DNA (27). DNAs were mixed with the cells and
subjected to 250 V and 960 µF with a Bio-Rad gene pulser. The
transfected cells were cultivated in 10-cm plates and harvested after
48 h. The cells were lysed by an alkali lysis method
(37) and centrifuged, and the low-molecular-weight DNA was
precipitated by addition of 0.7 volume of isopropanol to the supernatant. The pellets were dissolved in 200 µl of a buffer containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM EDTA, 0.2% sodium dodecyl sulfate (SDS), and 100 µg of proteinase K per ml and
incubated for 2 h at 37°C. After treatment with
phenol-chloroform-isoamyl alcohol and ethanol precipitation, the
pelleted DNA was washed with 70% ethanol and dissolved in TE buffer
(10 mM Tris-HCl [pH 8.0], 1 mM EDTA). To linearize the DNA, the
samples were digested with 10 U of EcoRI for HPV-16 and 10 U
of HpaI for HPV-31 and concomitantly with 10 U of
DpnI to cut the originally transfected, bacterially
replicated, and therefore methylated DNA. The restriction digests were
incubated for 5 h at 37°C, and the DNA was purified by standard
procedures, run on 0.8% agarose gels, and blotted onto a Hybond-N
nylon membrane. After the membrane was baked at 80°C for 2 h,
32P-end-labelled probes derived from HPV-16, HPV-31, and
BPV-1, respectively, were hybridized against the blotted DNA. The
membrane was washed in 0.1× SSC (1× SSC is 0.15 M NaCl plus 0.015 M
sodium citrate)-10% SDS for 20 min at 65°C and autoradiographed.
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RESULTS |
Sequences resembling the HPV-16 PSM are present between the
enhancers and promoters of all genital HPV types and bind the same
cellular factor.
All genital HPV types appear to have an
epithelial-cell-specific enhancer (14, 18, 23, 24, 32, 50)
that is flanked by two E2 binding sites, roughly 150 and 550 bp
upstream of the E6 promoter (for overviews, see references
44 and 50). The E6 promoter is
modulated by an Sp1 site and two further E2 binding sites (16,
59). The 60-bp region upstream of the Sp1 site toward the
enhancer is very AT rich in all genital HPVs (41). A
25-bp segment within this AT-rich segment of HPV-16 represses epithelial-cell enhancer-dependent transcription from the E6
promoter (45). We refer to this segment as PSM and to the
protein that binds and functionally activates a direct repeat of the
sequence 5'-TAYAATAAT-3', within this segment, as PSM-BP.
The 60-bp AT-rich region and its two flanking E2 binding sites seem to
be the replication origin of all papillomaviruses, as was shown for
BPV-1 (26, 39, 40, 61, 62), HPV-11 (31, 49),
HPV-16 (20), HPV-18 (57), and HPV-31 (21,
22, 27). Replication is initiated by the E2 protein bound to
either of the two flanking binding sites (22, 35, 54, 57)
directing the E1 replication initiation protein to an AT-rich 18-mer
binding site (26; for an alignment, see reference
44). Figure 1
summarizes these elements of HPV-16, and shows that the E1 binding site
overlaps with the PSM.
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FIG. 1.
Location of the PSM, the overlapping binding sites of
the replication protein E1, and other major binding sites for
transcriptional regulators in the LCR of HPV-16. The identification of
the nuclear MAR is described in reference 60, the
enhancer and its elements are described in references 4, 11,
13, 14, 23, and 44, the silencer elements
are described in references 38, 45, and
46, the replication origin is described in reference
15, and the promoter elements are described in
references 16 and 59.
Abbreviations: L1, L1 gene; E6, E6 gene; NFI, nuclear factor 1; GR,
glucocorticoid receptor; AP1, activator protein 1; TF1, transcription
enhancer factor 1; oct, octamer binding factor 1.
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An examination of this genomic region of many genital HPV types reveals
sequences bearing similarity to the two 5'-TAYAATAAT-3'
repeats of the HPV-16 PSM (
41). To determine whether
these sequences
could bind the same cellular factor, PSM-BP, as HPV-16,
we analyzed
sequences from a number of genital HPVs, namely, those from
HPV-2,
HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, and HPV-45 (Fig.
2). Figure
3A depicts the results of an EMSA in
which double-stranded oligonucleotides
containing the sequences shown
in Fig.
2 were radiolabelled and
used as probes for HeLa nuclear
extract. The characteristic low-mobility
complexes (denoted C1 and C2)
observed with the HPV-16 probe were
seen with all six other HPV
sequences. Based on a mutational analysis
of HPV-16, we had proposed
that C1 may represent a monomer of
PSM-BP complexed with one
5'-TAYAATAAT-3' submotif and that C2
may represent a PSM-BP
dimer, which binds efficiently to the 5'-TAYAATAAT-3'
repeat
within PSM but may form inefficiently on singular binding
sites. It
cannot be excluded, however, that C2 represents a heterodimer.
Several
fast-migrating bands most probably stem from nonspecifically
binding
proteins, while one additional complex, X, is barely visible
in HPV-16
and some other types but appears as a strong band in
HPV-18, HPV-11,
and HPV-2. The nature of the protein that gives
rise to complex X is
not known (see below).
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FIG. 2.
Alignments of presumed and known binding sites for the
replication factor E1 in seven genital HPV types and in BPV-1 coincide
with binding sites for the transcriptional repressor PSM-BP (or
CDP/Cut, respectively). The alignment of E1 binding sites was done as
in a study by Holt and Wilson (26) extended by a sequence
comparison (44). A bracket indicates the presumed E1
consensus binding sequence. Bold letters identify the PSM of HPV-16 and
similarities in six other HPV types. Since CDP/Cut binding sites do not
show a strict consensus sequence, with multiple 5'-TAAT-3'
5'-CAAT-3' motifs at variable positions and orientations
being the hallmark of the generally AT-rich sequences, the
identification of CDP/Cut targets in these PSMs is conjectural. The
mutations (underlined) were aimed at eliminating all of the
5'-TAAT-3' and most of the 5'-CAAT-3' elements.
All of these sequences overlap with the position 1 of the circular
genomic map. The exact genomic position is shown on the right. The
sequences shown represent oligonucleotides that were examined by EMSA
and cloned in the form of BamHI-BglII fragments
into the CAT expression vector p80SV for functional tests.
Oligonucleotides that were inserted in the sense orientation in this
vector were identified by DNA sequencing.
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FIG. 3.
The cellular factor PSM-BP binds to the homologous
genomic region of seven different HPV types. (A) EMSA studies with
probes containing PSM-like sequences (Fig. 2) including the origin of
replication of HPV-16, HPV-31, HPV-33, HPV-18, HPV-45, HPV-11, and
HPV-2 give rise to the same slow-migrating complexes C1 and C2
previously seen with the HPV-16 PSM. The nature of complex X, which is
barely visible in HPV-16 but strong in several other HPV types, is not
known. (B) The C1 and C2 complexes of HPV-31, HPV-18, HPV-11, and HPV-2
are competitively inhibited by an HPV-16 probe containing a wild-type
PSM (labelled 16 and consisting of the sequence
5'-CTAAACTACAATAATTCAT-3') but not by one containing
mutations within the PSM that inhibit PSM-BP binding (labelled m and
consisting of the sequence 5'-CTAAACTACACGCCGTCAT-3'). These
results suggest that the PSM-BP complexes C1 and C2 that form with the
sequences of these four HPV types are the same complexes previously
described for HPV-16 (45).
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Five of these seven HPV types were studied further in competition
experiments, and Fig.
3B shows that formation of the C1
and C2
complexes could be successfully inhibited by an oligonucleotide
with
the wild-type HPV-16 5'-TACAATAAT-3' sequence (labelled 16)
but not by a mutant HPV-16 sequence, 5'-TACACGCCG-3', that
fails
to bind PSM-BP (labelled m). We conclude from this that the seven
studied HPV types (and, as sequence inspection suggests, probably
all
genital HPVs) have motifs with similar PSM-BP binding
capabilities.
PSMs from different genital HPV types function as transcriptional
repressors.
We published recently the finding that the contiguous
LCR of many different HPV types has a dramatically lower
transcriptional activity than chimeric constructs in which the
enhancers of the same viruses are cloned in front of a canonical
Sp1-activated HPV E6 promoter (50). It became clear to us
that these two groups of constructs differed from one another by the
absence in the latter group of the 60-bp AT-rich segments containing
potential PSM elements. This strongly suggests the presence of a
silencer in these AT-rich segments of each of these HPV types. To
verify this and to confirm that the PSM is the element responsible for silencing the contiguous LCRs, we decided to compare the potential PSMs
of different HPV types in standardized test constructs.
We had previously observed that the HPV-16 PSM had similar silencing
activity whether it was positioned between the homologous
HPV-16
enhancer and promoter or when it was tested in the context
of
heterologous elements such as the SV40 enhancer (
45). To
investigate whether the potential PSMs from the HPV-2, HPV-11,
HPV-18, HPV-31, HPV-33, and HPV-45 would indeed have a silencing
activity similar to that of HPV-16, we cloned the sequences used
in the
EMSA experiments into the p80SV CAT reporter plasmid between
the SV40
enhancer and the HPV-16 E6 promoter. Figure
4A shows
the outcome of CAT assays with
extracts of C33A cells transiently
transfected with these six
constructs and control plasmids. As
seen in the figure, the SV40
enhancer strongly activates the HPV-16
promoter in construct p80SV
compared with the promoter activity
alone for construct p80. This
activity is almost completely repressed
by the presence of the HPV-16
PSM in p80SV16 but not by a mutant
sequence of the PSM in p80SV16m
(Fig.
2). The repressing effect
of the sequences of all of the six
other HPV types is similar
to that of HPV-16, identifying them as
functional PSMs.
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FIG. 4.
The PSM-like elements of seven different HPV types
function as strong transcriptional repressors. (A) As previously
described (45), the HPV-16 sequences containing a functional
PSM repress the activity of the heterologous SV40 enhancer (p80SV16)
while corresponding sequences containing mutations within the
5'-TAYAATAAT-3' motifs fail to do so (p80SV16m). Likewise,
the PSM-like sequences of six other HPV types (HPV-31, HPV-33, HPV-18,
HPV-45, HPV-11, and HPV-2) repress CAT expression comparably. (B)
Mutation of the PSM sequences of HPV-16, HPV-18, and HPV-31 (Fig. 2)
releases the repression of the SV40 enhancer-HPV-16 promoter chimera.
(C) When tested in EMSA in form of free oligonucleotides, the same
mutations as functionally tested in the experiment in panel B fail to
give rise to the characteristic C1 and C2 complexes of PSM-BP. The
nature of complex X, which is barely visible with the HPV-16 wild type
but strong in several other HPV types and in the HPV-16 mutant, is not
known. The fastest-migrating complex above the free probe (FP), which
is visible with wild-type sequences but absent with mutants, is likely
to be a degradation product of the C1/C2 protein.
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We had observed that the deletion of one of the two
5'-TAYAATAAT-3' repeats in HPV-16 led to a partial release
of repression.
Here we have examined the functional effect of multiple
point
mutations that altered most of the 5'-TAAT-3' and
5'-CAAT-3' motifs
within the PSMs. Figure
4B shows that
these mutations of the PSMs
of HPV-16, HPV-18, and HPV-31 (Fig.
2)
released the repressor
activity of the PSMs. The wild-type and mutant
HPV-16 vectors
used in the experiments in Fig.
4A and B are identical,
but the
two panels were derived from separate transfections. Figure
4C
shows that these mutations no longer allow the formation of complexes
C1 and C2 on the free oligonucleotides. The faster-migrating complex
X
is not affected by these mutations in HPV-18 and HPV-31 but
increases
in amount in HPV-16. We do not believe that the factor
giving rise to
complex X has any stimulatory effect on the transcription
of these
constructs, since deletion of the whole HPV-16 PSM segment
(including
the binding site for X) (
45) has the same effect
as these
point mutations that increase complex X formation. Together,
these
results suggest that the functional organization of the
PSMs of
different HPV types is
conserved.
The cellular factor PSM-BP is identical to the
differentiation-specific transcriptional repressor CDP/Cut.
Since
our findings had confirmed the PSM-BP as an important transcriptional
regulator of genital HPVs with a binding site overlapping the
replication origin, we were interested in identifying and
characterizing this factor. Figure 5
documents our attempts to purify PSM-BP from crude HeLa nuclear
extract. The first step involved a heparin-Sepharose ion-exchange
column. After binding of HeLa nuclear extract, maximal PSM-BP activity
eluted in the 0.3 M KCl fraction (Fig. 5A). A second enrichment was
achieved by precipitation of the proteins in this fraction by using a
fractionated ammonium sulfate precipitation and screening by EMSA for
PSM-BP activity. Maximal PSM-BP activity was observed in the 20 to 40% fraction (Fig. 5B). This fraction was further purified on a
Sephacryl-S400 column by gel filtration (Fig. 5C). A series of
molecular mass markers was used to provide an indication of the size of
PSM-BP, and it can be seen from Fig. 5C that it elutes close to the
position of the -amylase protein, which has a molecular mass of
approximately 200 kDa.
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FIG. 5.
The purification profile of PSM-BP, as monitored by
EMSA, is similar to that of the differentiation-specific factor
CDP/Cut. (A) Ion-exchange chromatography purification of HeLa nuclear
extracts on a heparin-Sepharose column demonstrates that PSM-BP binding
activity is maximal in the 0.3 M KCl fraction. FT, flowthrough. (B)
Ammonium sulfate precipitation of PSM-BP-enriched fractions of the
heparin-Sepharose chromatography defines PSM-BP 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 PSM-BP is a very large protein of approximately 180 kDa (as
defined by the -amylase molecular mass marker). V0, void
volume. The purification profile of PSM-BP presented here is consistent
with that described for the differentiation-specific transcriptional
repressor CDP/Cut (42). FP, free probe.
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We considered the possibility that PSM-BP is a previously known
transcription factor. Only a few repressors have been characterized
so
far from human and mammalian cells, and one of them, the CCAAT
displacement protein (CDP/Cut), is a large protein with a size
similar
to that observed for PSM-BP. CDP/Cut consists of 1,505
amino acid
residues, with a calculated molecular mass of 165 kDa,
and migrates in
SDS-polyacrylamide gel electrophoresis with an
apparent molecular mass
of 180 to 190 kDa. Moreover, CDP/Cut demonstrates
a purification
profile similar to that of PSM-BP (
42) and has
a propensity
for binding AT-rich sequences (
3).
To determine whether PSM-BP and CDP/Cut were the same
protein, we carried out a series of EMSA studies including
competition
experiments with different regulatory sequences that have
previously
been shown to interact with CDP/Cut. Figure
6A shows that sequences
from the
phagocyte-specific cytochrome heavy-chain gene promoter,
a
well-characterized binding site of CDP/Cut, give rise to complexes
with
identical mobilities to the HPV-16 PSM-BP complexes C1 and
C2. The fact
that the C2 complex is weaker than C1 in the gp91-phox
site may stem
from the fact that formation of a potential dimer
occurs less
efficiently on this singular binding site than on
the dimeric binding
sites of HPV-16. In competition experiments,
the gp91-phox probe and
another well-known CDP/Cut binding site
from a
Psammechinus
miliaris histone H2B promoter (
8) competitively
inhibit
the formation of C1 and C2 on the HPV-16 probe. Further
evidence
suggesting that PSM-BP is CDP/Cut is presented in Fig.
6B, where a
specific anti-CDP/Cut polyclonal antibody results
in the loss of C1 and
C2 complexes from the HPV-16 probe while
a preimmune serum fails to
prevent C1 and C2 formation. From the
combination of these data, we
conclude that PSM-BP is identical
to CDP/Cut.
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FIG. 6.
The repressor PSM-BP of HPV transcription is identical
to CDP/Cut. (A) An EMSA shows similarity of the complexes C1 and C2
generated by an HPV-16 PSM oligonucleotide and an oligonucleotide
representing a known CDP/Cut binding site of the phagocyte-specific
cytochrome heavy-chain gene gp91-phox. As outlined in reference
45, we interpret the higher intensity of C2 in the
left lane as a reflection of a dimer binding the two repeats of PSM,
while only a partially dimeric CDP/Cut factor population binds the
singular binding site of gp91-phox. The strong complex with higher
mobility in the gp91-phox lane reflects the binding of the CCAAT
binding protein CP1, and the fast-migrating complex in both lanes is a
possible CDP/Cut degradation product. The next three lanes show
competition for complex formation by a homologous probe (lane HPV-16)
and probes containing CDP/Cut binding sequences from the gp91-phox gene
and the P. miliaris histone H2B gene. By contrast, a probe
containing a mutated PSM only slightly interferes with C1 and C2
formation (right lane). CP1 identifies the complex formed on the
gp91-phox promoter with the CCAAT binding protein. Bands that migrated
faster than CP1 are degradation products of CDP/Cut. FP, free probe (B)
An anti-CDP-specific antibody ( -CDP) abolishes the formation of
HPV-16 PSM-BP complexes C1 and C2. This effect is specific and is not
seen upon the addition of a preimmune serum (PI) to the EMSA
reaction.
|
|
Repression of HPV-16 transcription by CDP/Cut involves a
histone-mediated mechanism.
CDP/Cut represses transcription by two
alternative mechanisms, displacement of activators (8, 36,
63) and binding of the histone deacetylase HDAC1, whose activity
changes nucleosomes such that it becomes difficult for the
transcriptional machinery to access the DNA (33). For
genital HPVs, this means either that the PSMs may bind an activator
that is displaced by CDP/Cut or that HDAC1 bound to CDP/Cut may modify
two nucleosomes that overlap (at least in HPV-16 and HPV-18) with the
enhancer and the E6 promoter (56) such that these elements
become inaccessible to transcription factors. We considered the second
possibility more likely, since PSM deletion mutants of HPV-16, which
would also delete the binding site of a potential activator, are
strongly upregulated (45).
To examine this possibility, we transiently transfected HeLa cells with
vectors containing the natural (EP-Luc) and mutated
(E*P-Luc) CDP/Cut
binding sites (Fig.
2) of HPV-16 and treated
the cells with the HDAC1
inhibitor TSA. These luciferase test
vectors contained the HPV-16
enhancer and promoter based on a
cassette system used in the context of
the CAT reporter gene (
45).
This analysis is complicated by
the fact that the HPV-16 silencer
contains five binding sites for
the YY1 factor, which is, just
like CDP/Cut, also known to
interact with a histone deacetylase
(
65). Figure
7 shows the outcome of this experiment.
Expression
of luciferase from EP-Luc, containing two binding sites for
CDP/Cut
and five sites for YY1, is stimulated by TSA 9-fold, probably
due to the combined effect of TSA on the histone deacetylase
complexed
with both factors. Mutation of the two CDP/Cut
sites in E*P-Luc
leads to a threefold increase of the uninduced level
and, through
a further 2.5-fold stimulation by TSA, to a total
expression similar
to that of the TSA stimulated EP-Luc. In the absence
of CDP/Cut
binding sites, this stimulation should stem from
interference
with the YY1-associated histone deacetylase. From this we
conclude
that the ninefold TSA stimulation of EP-Luc contains two
components,
namely, the 2.5-fold stimulation still detectable with
E*P-Luc
due to YY1, and a further 3- to 4-fold stimulation due to the
CDP/Cut associated HDAC1. This amount should be identical to the
difference between the uninduced EP-Luc and E*P-Luc vector. These
observations indicate that CDP/Cut bound to PSM represses by modifying
the nucleosomes established on the HPV LCR.
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FIG. 7.
Repression of HPV-16 transcription by CDP/Cut involves a
histone-mediated mechanism. Luciferase expression in HeLa cells
transiently transfected with vectors containing the natural (EP-Luc)
and mutated (E*P-Luc) CDP/Cut binding sites of HPV-16 is stimulated by
treatment with the HDAC1 inhibitor TSA.
|
|
Overexpression of the CDP/Cut cDNA represses replication of HPV-16,
HPV-31, and BPV-1.
The presumed PSMs of all genital HPVs are found
in the same genomic region, overlapping with position 1 of the
nucleotide sequence of the respective genome (Fig. 2). As determined
for a number of HPV types (20, 21, 27, 31, 49, 57) and presumed for a number of others, this region represents the binding sites of the replication factor E1 (for an alignment of HPV replication origins, see reference 44). It is therefore
distinctly possible that E1 and CDP/Cut sites overlap, and it presents
the interesting possibility that CDP/Cut also regulates HPV replication.
To test this possibility, we studied the effect of overexpressing
CDP/Cut on HPV replication in vivo. Toward this end, we
used both the
HPV-16 replication origin present within the complete
LCR cloned into a
bacterial plasmid and the complete recircularized
and plasmid-free
HPV-31 genome. As recipient cells, we used the
cell line Cho4.15, which
has been engineered to express the BPV-1
proteins E1 and E2 under the
influence of heterologous promoters
from intrachromosomally recombined
genes. These proteins are known
to activate heterologous HPV
replication origins (
15,
48).
Figure
8 shows that the HPV-16 LCR
construct was indeed able to replicate in these cells (lane 1).
Cotransfection of the empty
expression vector pMT2 (
42) had
no effect on HPV-16 replication
(lane 2). By contrast, coexpression of
a pMT2-CDP construct that
contains the full-length CDP/Cut cDNA
abolished HPV-16 replication
(lane 3). This effect was not limited to
HPV-16, since replication
of HPV-31 was also repressed in a
dose-dependent manner by the
overexpression of CDP/Cut (compare lanes 5 and 6 with lane 4).
These data and the intriguing overlap of the E1 and
the CDP/Cut
binding sites suggest that CDP/Cut can repress HPV
replication
and that CDP/Cut simultaneously regulates both
transcription and
replication.
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FIG. 8.
CDP/Cut inhibits transient HPV replication in vivo. Cho
cells stably expressing E1 and E2 proteins were transfected with
pBluescript SK(+) carrying the HPV-16 LCR or with recircularized HPV-31
DNA. Total cellular DNA was prepared after 48 h, digested with
DpnI to remove the input bacterially replicated DNA,
linearized with EcoRI (HPV-16) or HpaI (HPV-31),
respectively, blotted, and hybridized with an LCR fragment of HPV-16
(lanes 1 to 3) or of HPV-31 (lanes 4 to 6). The arrow denotes the
position of the linearized replication products, which have molecular
sizes of 4 kb in pBluescript SK(+) HPV-16LCR (arrow in the left panel)
and 7.9 kb in the linearized HPV-31 genome (arrow in the right panel).
Cotransfection of the CDP expression vector pMT2-CDP strongly represses
replication for both HPV types (compare lane 1 with lane 3, and lanes 4 with lanes 5 and 6. The expression vector pMT2 without CDP sequences
does not interfere with the replication of HPV-16 (lane 2).
|
|
As an alternative to this interpretation, one could suspect that
CDP/Cut influences the expression of E1 and E2 from the heterologous
promoters used in Cho4.15 with the downstream consequence of repression
of replication. To show that CDP/Cut represses replication
independently
from such a potentially artifactual system and to extend
our study
to a papillomavirus type unrelated to the genital HPVs, we
also
examined the replication of BPV-1. Figure
2 shows that this virus
contains potential CDP/Cut binding sites that overlap with the
binding
site of the E1 protein in a manner similar to the arrangement
of
sequences in genital HPVs. EMSA results in Fig.
9A show that
these homologous sequences
indeed lead to band shifts indistinguishable
from those with HPV-16 and
HPV-2 sequences. To study the effect
of CDP/Cut on the replication of
BPV-1, we transfected ID13 fibroblasts,
a mouse c127 cell-derived line
with about 100 stably episomally
replicating BPV-1 genomes, with the
pMT2-CDP expression vector.
Figure
9B shows stepwise decreasing
concentrations of BPV-1 genomes
after application of 0.5, 5, and 12 µg of pMT2-CDP (slots 2 plus
3, 4 plus 5, and 6 plus 7, respectively)
in comparison with cells
transfected only with the empty expression
vector (slot 1). These
data prove that CDP/Cut interferes efficiently
with papillomavirus
replication, although this experiment does not
distinguish between
an interference with E1 function, as suggested by
us, and a potential
repression of the homologous E1 and E2 promoters of
BPV-1 with
the downstream consequence of repression of replication.
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FIG. 9.
(A) An oligonucleotide representing the replication
origin and presumed CDP/Cut binding sites of BPV-1 generates bandshifts
that are indistinguishable from those of HPV-16 and HPV-2. (B)
Transfection of ID13 cells, which contain about 100 episomally
replicating copies of BPV-1, with increasing amounts of the CDP/Cut
expression vector pMT2-CDP, decreases the copy number of BPV-1. The
cells were electroporated with 4 µg of the empty vector (slot 1), 0.5 (slots 2 plus 3), 4 (slots 4 plus 5), and 12 µg (slots 6 plus 7) of
pMT2-CDP and grown for 48 h. Then DNA was harvested, cleaved with
HindIII, and processed with a radioactive probe for
BPV-1 DNA. The arrow points to the 8-kb band generated by the
linearized BPV-1 genome.
|
|
| |
DISCUSSION |
The repressor CDP/Cut binds a nucleotide sequence that is conserved
among many HPV types.
We have reported here five lines of evidence
suggesting that PSM-BP is identical to the transcription factor
CDP/Cut: (i) PSM-BP and CDP/Cut are both transcriptional repressors
with a propensity for binding AT-rich sequences, (ii) PSM-BP and
CDP/Cut have identical purification profiles and similar molecular
weight, (iii) the known CDP/Cut binding site on gp91-phox gives rise to complexes in EMSA studies that have identical mobilities to the HPV-16
PSM-BP C1 and C2 complexes, (iv) formation of the HPV-16 C1 and C2
complexes on the PSM can be competitively inhibited by two known CDP
binding sites, and (v) a specific anti-CDP/Cut antibody abolishes the
formation of the C1 and C2 complexes in EMSA experiments with the
HPV-16 probe. Collectively, these data leave little doubt that the
transcriptional repressor termed PSM-BP in our previous study is, in
fact, CDP/Cut.
CDP was originally described from a study of a histone H2B gene from
the sea urchin
P. miliaris. This gene is selectively
expressed in sperm cells but repressed in embryonic cells. Activation
of the H2B promoter in sperm cells depends on the complex formation
between a CCAAT motif and the CCAAT binding factor, CP1. However,
formation of this complex is abrogated in embryonic cells by CDP,
which
binds sequences overlapping the CCAAT motif, thus blocking
DNA binding
by CP1 (
8). CDP was consequently named to reflect
this
initial observation. Later, it was found that CDP can repress
by two
distinct mechanisms, factor displacement (
8,
53) and
the
function of a silencer domain without displacing an activating
transcription factor (
36,
63). The recent finding that
CDP/Cut
associates with the histone deacetylase HDAC1 provides a
potential
explanation for this second activity, namely, structural
alteration
of nucleosomes (
33). Our observation of the
stimulation of HPV-16
transcription by TSA suggests that this mechanism
is active in
genital
HPVs.
CDP was found to be a homologue of the
Drosophila Cut
homeodomain protein, which plays a role in the determination of cell
fate in various tissues (reference
42 and references
therein);
it is now frequently referred to by the combined abbreviation
CDP/Cut. As in
Drosophila, CDP is believed to be a general
repressor
of developmentally regulated genes in mammals. Examples
include
regulation of the cytochrome heavy-chain gene gp91-phox during
myeloid differentiation (
34,
53) and expression of the
immunoglobulin
heavy chain during B-cell differentiation
(
63).
CDP/Cut recognizes defined binding sites, as judged, for example, by
footprint analysis, but it does not specifically recognize
CCAAT boxes
or any other clearly recognizable and conserved sequence
motif. Natural
and artificial CDP/Cut binding sites are AT rich
and often contain the
sequences 5'-TAAT-3' and 5'-CAAT-3' repeated
directly or invertedly with variable spacing (
3). Lack of
specificity
is in part explained by the finding that the CDP/Cut
contains
four DNA binding domains with different but overlapping
sequence
specificity (
6,
25). Multiple 5'-TAAT-3'
and 5'-CAAT-3' motifs
occur in each of the PSMs of the
seven genital HPV types studied
here.
PSM coupling of HPV transcription and epithelial
differentiation.
The activity of HPV enhancers and promoters
depends upon the cooperation between more than 10 different factors
(for a review, see reference 44). The specificity of
HPVs for epithelial cells is apparently dependent upon only a subset of
these factors, with most data available from studies of NF-I
(4), AP-1 (60), and Sp1 (5). These
three factors are each derived from multigene families, with the
epithelial-cell specificity originating from the fact that different
members of these gene families differ in function and are expressed in
a cell-type-specific manner. While these three factors appear to bind
the LCRs of all genital HPV types (44, 50), another factor,
skn-1, expressed exclusively in epithelial cells, has so far been found
to activate only HPV-1 and HPV-18 gene expression (2, 66).
Consequently, regulation by skn-1 does not provide an explanation for
the general phenomenon of epithelial-cell specificity of genital HPV enhancers.
It is probably the principal function of these three transcription
factors (NFI, AP-1, and Sp1) to activate HPV gene expression
selectively in an epithelial environment, leaving HPV genomes
dormant
in nonepithelial cells. However, other factors may regulate
transcription in a differentiation-dependent manner during
stratification.
Differential gene expression in the different layers of
epithelia
is well documented for both cellular (
30) and HPV
(
28,
55)
genes. The sequences contained within the E6
promoter may represent
one way by which HPVs modulate early
transcription, since the
relative concentration between the activator
Sp1 and its antagonist
Sp3 changes during epithelial differentiation
(
5).
Recent publications have provided new and exciting data suggesting that
CDP/Cut may represent another and quantitatively more
important factor
that couples HPV transcription to epithelial-cell
differentiation
(
1,
47). This is because the activity of
CDP/Cut strongly
decreases during epithelial-cell differentiation
and correlates with
increased transcription from three different
early promoters of HPV-6.
These findings suggest that stratified
epithelia are yet another
example where CDP/Cut influences the
developmental regulation of gene
expression. The modulation of
the HPV-6 E6 promoter observed by Roman
and colleagues (
1,
47) is probably derived from the HPV-6
PSM, since the promoter
fragment studied by these authors includes this
motif. Together,
these findings, along with the results presented here,
suggest
that CDP/Cut, by binding the conserved PSM of HPVs, may provide
an important regulatory link between epithelial differentiation
and HPV
early gene
expression.
Roman and colleagues also reported that other regions of the HPV-6
genome, namely, the 5' part of the LCR, the E6 gene, and
the E7 gene,
bind CDP/Cut (
1,
47), resulting in the repression
of the E6
promoter, the E7 promoter (which is absent from HPV-16
and most other
genital HPVs), and the E1 promoter. In our ongoing
research on the
nature of nuclear matrix attachment regions (MARs)
of HPVs
(
58), we have found that HPV-16 MARs also contain clusters
of CDP/Cut binding sites (W. Stünkel, M. J. O'Connor, and
H.
U. Bernard, unpublished results). The MARs of HPV-16 are
located
in the same genomic positions as the CDP/Cut binding regions of
HPV-6 (
1,
47) outside the HPV-6 E6 promoter fragment. The
CDP/Cut binding regions of HPV-6 are, by sequence analysis, likely
to
be MARs (
58). CDP/Cut has been proposed to be attached the
nuclear matrix (
7), suggesting some functional relationship
between MARs and clusters of CDP/Cut binding sites. While details
of
these mechanisms await further research, it is apparent that
in
addition to the important role played by the PSM of each HPV
type,
there are, in the form of MARs, conserved silencing elements
within HPV
genomes with CDP/Cut binding sites. The PSM and MARs
together may form
a complex network of cooperations among one
another and with other
cellular components to achieve synchronization
of HPV gene regulation
and epithelial
differentiation.
CDP/Cut represses papillomavirus replication and may couple this
process to epithelial-cell differentiation.
HPV genomes are much
more abundant in supraepithelial cells than in basal cells
(43), and their efficiency of replication apparently depends
on epithelial-cell differentiation (10). There are a number
of observations suggesting that CDP/Cut binding to the PSM of HPVs
could be partly or wholly responsible for the differentiation-specific
regulation of HPV replication. These include (i) the overlapping of the
PSM (and therefore the CDP/Cut binding sites) with the HPV E1 binding
site, (ii) the repression of HPV-16 and HPV-31 replication by
exogenously expressed CDP/Cut protein, and (iii) as described above,
the differentiation-specific expression of the CDP/Cut protein itself.
While we have provided the first examples of how CDP/Cut can repress
papillomavirus replication in vivo, it is not clear exactly
what
mechanism is involved. One obvious possibility is that CDP/Cut
prevents
E1 from binding to its cognate sites within the origin.
However, E1
binding to sequences within the origin is a complex
affair in which
active E1 complexes depend upon the correct multimerization
of E1
proteins (
52). Moreover, E1 has the potential to bind
to
alternative sites within the AT-rich sequences that include
the origin,
although binding to these sites might not necessarily
give rise to
active E1 complexes (A. Stenlund, personal communication).
Thus,
CDP/Cut binding to the PSM might not prevent the binding
of E1 to the
origin of replication but might prevent the formation
of an active E1
complex. Another nonexclusive mechanism could
be invoked that involves
the association of histone deacetylase
activity with CDP/Cut
(
33). In such a mechanism, deacetylation
of the histones of
nucleosomes positioned in the vicinity of the
replication origin
(
56) could result in a chromatin structure
that prevents
active replication. The need for nucleosomal alterations
during the
initiation of papillomavirus replication has recently
been highlighted
by the finding that E1 interacts with components
of the Swi/Snf complex
(
32b). Future studies should provide insight
into these
possible
mechanisms.
In summary, we have provided evidence that the PSM originally
identified in HPV-16 is conserved in all genital HPVs tested.
The
factor that binds to the PSM has now been identified as the
differentiation-specific repressor CDP/Cut. What is more, the
conservation of positioning within the origin of replication does
not
appear to be a coincidence, and we provide evidence that in
addition to
transcriptional repression, CDP can repress HPV replication
in vivo.
Thus, through one regulatory motif, both HPV early-gene
expression and
replication may be coupled to the differentiation
program of the host
stratified
epithelia.
| |
ACKNOWLEDGMENTS |
M.J.O. and W.S. contributed equally to this work.
We thank S. H. Orkin and E. Neufeld for plasmids pMT2 and pMT2-CDP
and anti-CDP antiserum, W. G. Hubert and L. A. Laimins for
pBR322-HPV-31, and M. Ustav and I. Ilves for the cell line Cho 4.15 (E1/E2).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Molecular and Cell Biology, 30 Medical Dr., Singapore 117 609, Singapore. Phone: (65) 8743765. Fax: (65) 7791117. E-mail:
mcbhub{at}imcb.nus.edu.sg.
Present address: KuDOS Pharmaceuticals Ltd., Cambridge CB4 4GW,
United Kingdom.
| |
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Abstract
Introduction
