Download Identification and characterization of a cluster of transcription start

Journal of General Virology (2003), 84, 2909–2920
DOI 10.1099/vir.0.19332-0
Identification and characterization of a cluster of
transcription start sites located in the E6 ORF of
human papillomavirus type 16
Maiken W. Rosenstierne, Jeppe Vinther, Christina N. Hansen,
Martin Prydsoe and Bodil Norrild
Correspondence
Bodil Norrild
[email protected]
Received 6 May 2003
Accepted 8 August 2003
Institute of Molecular Pathology, The Protein Laboratory, University of Copenhagen, Panum
Institute, Bldg 6.2, Blegdamsvej 3C, DK.2200 Copenhagen N., Denmark
Human papillomavirus type 16 (HPV-16) is the prototype strain among the malignant types of HPV
in the western world. The main promoter, P97, located in front of the E6 ORF, has been
shown to control expression of the oncogenes E6 and E7. These oncogenes are expressed
continuously in HPV-16-transformed cells. In contrast to malignant HPV types, non-malignant HPV
types have separate promoters driving the expression of E6 and E7. Experiments have shown
that the translation of E7 is more efficient from monocistronic than bicistronic transcripts encoding
both E6 and E7. Here, identification of a cluster of transcription start sites located in the E6
ORF of HPV-16 is presented. Transcripts from this region contain the E7 ORF as the first reading
frame. The cluster consists of multiple transcription start sites located around nt 441.
Additional transcription start sites were identified in a cluster around nt 480. A transcription start site
has been identified previously at nt 480 but has never been characterized further. The region
responsible for transcription activity was mapped to nt 272–448. Mutational analysis showed that
initiation of transcription is independent of a TATA-box element, which is consistent with the
finding of multiple transcription start sites. Furthermore, it is shown that proteins from HeLa and
SiHa nuclear cell extracts bind to the two regions at nt 291–314 and 388–411, and that these two
regions influence transcription activity in a cell type-dependent manner.
INTRODUCTION
Human papillomaviruses (HPVs) are epitheliotropic DNA
viruses with a double-stranded circular genome of approximately 8 kbp. During the last 20 years, more than 85
different types of HPV have been identified and characterized, and novel types are still being isolated (zur Hausen,
1999). HPV types are divided into malignant and nonmalignant types according to their ability to transform the
host cell. The HPV genome consists of an early region
encoding six to seven early proteins [E6, E7, E1, E2, E4 (E8)
and E5], a late region encoding two late proteins (L1 and L2)
and two regulatory regions, designated the long control
region (LCR) and the small non-coding region (Seedorf
et al., 1985). In the western world, HPV-16 is considered to
be the prototype strain of the malignant types of HPV. The
oncoproteins E6, E7 and E5 from malignant types of HPV
are responsible for the transformation of the epithelial host
cell. In the normal life cycle of the virus, oncoproteins
induce unscheduled replication in differentiating keratinocytes to facilitate virus replication (for reviews, see zur
Published ahead of print on 27 August 2003 as DOI 10.1099/
vir.0.19332-0.
0001-9332 G 2003 SGM
Hausen, 1996; and Zwerschke & Jansen-Dürr, 2000). The
E1 and E2 proteins are responsible for DNA replication
(reviewed by Phelps et al., 1998) and E2 also functions as a
transcriptional regulator (Nishimura et al., 2000; Tan et al.,
1994; Ushikai et al., 1994). The E1^E4 protein binds to
intermediate filaments and is believed to facilitate the release
of virus particles. In addition, the E1^E4 protein is believed
to be involved in post-transcription regulation (Doorbar
et al., 2000). L1 and L2 are the capsid proteins (reviewed by
zur Hausen, 1996).
Transcription regulation of the HPV genome is very complex
and is controlled largely by the LCR, which binds cellular
transcription factors and the HPV E2 protein (reviewed by
O’Connor et al., 1995; for recent references, see O’Connor
et al., 1998, 2000; Stünkel & Bernard, 1999; and Stünkel
et al., 2000). Malignant HPV types have a main promoter
located in front of the E6 ORF. This promoter is named P97
in HPV-16 (transcription initiation at nt 97) (Smotkin et al.,
1989), P105 in HPV-18 (Thierry et al., 1987) and P99 in
HPV-31b (Ozbun & Meyers, 1998). These promoters all
produce differentially spliced polycistronic transcripts encoding the early genes. In contrast, non-malignant HPV types
have the main promoter located in the E6 ORF in front of
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M. W. Rosenstierne and others
the E7 ORF, namely P270 in HPV-6 (Smotkin et al., 1989)
and P264 in HPV-11 (Chow et al., 1987). Non-malignant
HPV types also have promoters in front of the E6 ORF and
these are responsible for the expression of E6, namely P80/
P90 in HPV-6 and P90 in HPV-11 (Karlen et al., 1996;
Smotkin et al., 1989; Zhao et al., 1997). A difference between
the malignant and non-malignant HPV types is therefore
the production of transcripts encoding the E7 protein as the
first ORF.
In eukaryotes, the translation of the second ORF in a polycistronic transcript is generally very inefficient and it has
been shown that the translation efficiency of the HPV-16
E7 oncoprotein is only 10 % compared to translation of a
monocistronic E7 transcript (Stacey et al., 1995). It is
possible therefore that a minor promoter that produces E7
transcripts as the first reading frame could contribute considerably to the overall expression of E7. In some malignant
types of HPV, multiple transcription start sites have been
identified in the E6 ORF but they have never been characterized. In HPV-18, transcription start sites were mapped to
nt 200, 215, 310 and 455 (Schneider-Gädicke & Schwarz,
1986). In HPV-16, a transcription start site was identified
in the E6 region at nt 480 but this was not characterized
further (Grassmann et al., 1996). We have previously
identified and characterized a new promoter, P542, with a
transcription start site at nt 542 in front of the E7 ORF
(Braunstein et al., 1999; Glahder et al., 2003).
Our previous work indicated the existence of promoter
activity located upstream of P542. This led us to search for
additional transcription start sites in the HPV-16 E6 ORF
that could contribute to the expression of E7. In this study,
we identify a novel cluster of transcription start sites mapped
to nt 430, 441 and 446. In addition, we find multiple transcription start sites clustered around the transcription start
site identified previously at nt 480 (Grassmann et al., 1996).
We have mapped the region responsible for transcription
activity to nt 272–448 and we show that several nuclear
proteins from the SiHa and HeLa cell lines bind to two
terminal sites at nt 291–314 and 388–411 of this region.
Furthermore, we find that the fragments spanning nt 272–
332 and 388–448 are important for the transcription activity
of the region at nt 272–448. We also show that transcription
activity is independent of the presence of a TATA-box-like
element.
METHODS
Cell culture. The HPV-negative cervical carcinoma cell line C33A,
the HPV-18-positive cervical cell line HeLa and the HPV-16-positive
cervical cell lines SiHa and CaSki were cultured in DMEM supplemented with 10 % FCS, 1 % L-glutamine and 1 % penicillin and
streptomycin at 37 uC and 5 % CO2. The day before transfection,
26105 cells were seeded in 3?5 cm 6-well Petri dishes (Nunc). Cells
were then transfected using LipofectAMINE Plus (Invitrogen) and
0?5 pmol DNA per dish, following the manufacturer’s instructions.
At 48 h post-transfection, cells were lysed with 300 ml Cell Culture
Lysis reagent (Promega) with shaking for 90 min. Lysates were then
used in a luciferase assay. Luciferin substrate (50 ml) was mixed
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manually with cell lysate (20 ml) and counted in a Turner 20/20
luminometer with a 3 s delay and a 12 s integration period. Each
construct was tested in at least four independent experiments in triplicates using two different preparations of DNA (a minimum of
12 measurements).
5§RACE PCR and cloning. The 59RACE protocol used in these
experiments is based on the CapFinder PCR cDNA principle
(Chenchik et al., 1998). Total RNA was purified using TRIzol
reagent (Invitrogen) and DNase treatment was performed with amplification grade DNase I (Invitrogen), according to the manufacturers’
instructions. mRNA purification was performed using the Oligotex
mRNA kit (Qiagen), according to the standard procedures of the
manufacturer. First-strand synthesis was performed using SuperScript II (Invitrogen). MnCl2 (2 or 3 mM) and BSA (1 mg ml21)
were added to 56 first-strand buffer (Schmidt & Mueller, 1999).
mRNA (100 ng) from the HPV-16-positive CaSki cell line was used
as template with a poly(dT) primer and a special cap primer
(rXhoICap, 59-aagcagtggtatcaacctcgagtacgc(ggg)r-39). First-strand
reactions were then treated with RNase H (Boehringer Mannheim),
according to the manufacturer’s instructions. PCR amplification of
first-strand cDNA was performed with Pfu polymerase (Promega)
using the programme: one cycle of 94 uC for 4 min, followed by five
cycles of 94 uC for 30 s, 66 uC for 1 min and 72 uC for 1 min, five
cycles of 94 uC for 30 s, 62 uC for 1 min and 72 uC for 1 min, and
20 cycles of 94 uC for 30 s, 58 uC for 1 min and 72 uC for 1 min.
The primers used were an HPV-16-specific primer (770bHindIII,
59-catggacaagctttgtacgcacaaccgaagcgtagagtcacac-39), containing an
HindIII restriction site, and the cap primer (XhoICap, 59-aagcagtggtatcaacctcgagtacgcggg-39), containing an XhoI site. cDNA was then
visualized on a 1?2 % agarose gel. A 2 ml sample of the PCR mixture
(100 ml) was used in a new PCR protocol using the programme: one
cycle of 94 uC for 4 min, followed by 20 cycles of 94 uC for 30 s,
58 uC for 1 min and 72 uC for 1 min. The cDNA was then inserted
into the pGL3-Basic vector (Promega) and sequenced.
Cloning of reporter constructs. The template used was the
pBR322 plasmid containing the HPV-16 genome (a gift from H. zur
Hausen, DKFZ, Heidelberg). Different fragments of the HPV-16
genome were amplified by PCR using Pfu polymerase and specific
primers containing restriction sites for KpnI (sense) and NheI (antisense). The coordinates of the different primers are nt 272–291,
332–352, 388–409, 448–463, 332–314, 388–370, 448–427 and 496–478.
The different fragments were inserted into the promoter- and
enhancerless pGL3-Basic or pGL3-Enhancer vectors (Promega), the
latter contains the simian virus 40 (SV40) enhancer. pGL3-Basic
constructs were all modified to reduce background from the vector,
as described by Braunstein et al. (1999). Site-directed mutagenesis of
the nt 272–448 fragment was performed using primers containing
two point mutations (59-gcatggacgctagccttttcttcaggacacagtggcttttgacagttaatacacctacgtaacaaatca-39 and 59-gcatggacgctagccttttcttcaggacacagtggcttttgacagtgaagacacctaatt-39; mutations are shown in bold).
The PCR product was amplified using Taq polymerase (Amersham
Pharmacia Biotech) to avoid 39R59 exonuclease proofreading activity. All constructs were sequenced using T7 Sequenase DNA polymerase, version 2.0 (Amersham), according to the manufacturer’s
instructions. Products were separated on a 6 % urea/polyacrylamide
gel and exposed to a BioMax MS X-ray film (Kodak).
RNase protection assay. Different probes were designed to span
the HPV-16 genome from nt 53 to 632 (probe F), 277 to 526 (probe
PI), 409 to 600 (probe PII) and 277 to 431 (probe PIII). The nt
53–632 fragment was amplified by PCR (59-atggacggatccgaaaccggttagtataaaagcagac-39 and 59-gtggacgaattccagtagagatcagttgtctc-39) and
inserted into the pAlter-1 vector using the BamHI/EcoRI restrictions
sites. The construct was then linearized with HindIII and used as
template in a T7 in vitro transcription reaction. The fragments spanning the regions nt 277–526, 409–600 and 277–431 were amplified
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HPV-16 and transcription
by PCR using the following primers: PI, 59-gcatggacctcgagtccatatgctgtatgtgataaatg-39 and 59-gcatggacggtacctctgcaacaagacatacatcgaccg-39;
PII, 59-gcatggacctcgaggtgtattaactgtcaaaagccactg-39 and 59-gcatggacggtacctaacatatattcatgcaatgtaggtg-39; PIII, 59-gcatggacctcgagtccatatgctgtatgtgataaatg-39 and 59-cgttaggtaccgtggcttttgacagttaatacacc-39. PCR
products were then inserted into the Bluescript IISK+ vector using
the KpnI/XhoI restriction sites. These constructs were all linearized
with BamHI and also used as templates in a T7 in vitro transcription
reaction. In vitro transcription was performed with MAXIscript
(Ambion) in the presence of [a-32P]UTP to create labelled antisense
probes. The probes were gel-purified and used in RNase protection
assays (RPA III, Ambion). Probe F (50 000 c.p.m.) was mixed with
5 and 10 mg CaSki RNA. Probes PII (87 000 c.p.m.) and PI and PIII
(80 000 c.p.m.) were mixed with 10 mg CaSki RNA. Prior to the assay,
CaSki RNA was DNase-treated, according to the manufacturer’s
instructions (Invitrogen). Unspecific RNA was degraded by digestion
with RNase A/T1 (diluted 1 : 50 or 1 : 25) according to the protocol.
Protected fragments were separated on a denaturing 5 % (7?5 % for
PI and PIII) polyacylamide gel run at 260 V and visualized by
exposing the gel to film (BioMax MS) for a minimum of 72 h. The
RNA Century Marker Template set (Ambion) was used according to
the manufacturer’s instructions to generate the RNA marker.
Electrophoretic mobility shift assay (EMSA). The regions span-
ning nt 272–448, 272–332, 332–388 and 388–448 of the HPV-16
genome were amplified by PCR and inserted into the pGL3-Basic
vector using the NheI restriction site. Constructs were cut with NheI
to make probes with a 59 overhang. Probes were purified using 1 %
agarose gels, according to the manufacturer’s instructions (Qiagen).
Probes were labelled with [a-32P]dATP using Klenow (New England
Biolabs) and purified using Sephadex columns (Boehringer
Mannheim). Small competitors were obtained by annealing complementary oligonucleotides (DNA technology, Århus). In each reaction, 10 mg HeLaScribe nuclear cell extract (Promega) or 15 mg SiHa
nuclear cell extract, purified according to the procedure described by
Dignam et al. (1983), were used together with 0?1 pmol probe ml21.
Competitor DNA was added in 10-, 25- and 50-fold concentrations
of the probe. Reactions were separated on a 6 % polyacrylamide gel
and visualized by exposing the gel to BioMax MS film over night.
RESULTS
Novel transcription start sites in the HPV-16
genome
To identify novel potential transcription start sites in the E6
ORF of HPV-16, 59RACE was used with mRNA isolated
from the CaSki cell line. The method is based on Clontech
CapFinder PCR cDNA synthesis (Chenchik et al., 1998).
MnCl2 was included in first-strand cDNA synthesis in order
to eliminate amplification of pre-terminated transcripts or
partially degraded transcripts (Schmidt & Mueller, 1999).
cDNA was amplified by PCR with an HPV-16-specific
primer (770bHindIII) and the cap primer (XhoICap)
(Fig. 1A). PCR products were analysed by agarose gel
electrophoresis; several products corresponding to different
59 ends were observed (Fig. 1B). cDNAs were inserted into
the pGL3-Basic vector using the HindIII/XhoI restriction
sites. Colonies were then sequenced. The 59 ends for the
main promoter P97 were mapped to nt 95 (data not shown)
and 97 (Fig. 1C). Two 59 ends were mapped to nt 483 and
489. Promoter P542, identified previously, was confirmed
http://vir.sgmjournals.org
also. In addition, three 59 ends were mapped to nt 430, 441
and 446.
To verify the novel 59 ends, RNase protection studies were
performed with total RNA extracted from CaSki cells. A long
antisense probe spanning the region of nt 53–632 (probe F)
of the HPV-16 genome was hybridized with different
amounts of CaSki RNA. After hybridization, unprotected
RNA was degraded by digestion with RNase A/T1 and
protected fragments were separated in a polyacrylamide gel.
Protected fragments corresponding to transcripts originating from the main promoter (P97) with the known splice
sites at nt 226^409 and 226^526 (Doorbar et al., 1990) were
detected (Fig. 2A, B). In addition, two protected fragments
of approximately 150 and 185 bp were observed. The
experiment was performed several times with different RNA
batches of CaSki and W12 cells (data not shown); these
fragments were observed repeatedly (Fig. 2A, B). These two
fragments could correspond to transcripts initiating around
nt 441 and 480, respectively. In addition, a fragment of
approximately 260 bp was observed repeatedly; the identity
of this fragment is still unknown.
To verify that the fragments observed were specific and
originated from the 39 end of probe F, we repeated the
RNase protection assay, this time using three small overlapping probes spanning the regions of nt 277–526 (PI),
409–600 (PII) and 277–431 (PIII) (Fig. 2C–E). Three protected fragments, which could correspond to transcripts
originating from P97, nt 441 and 430, were observed using
either PI or PII, but not PIII (Fig. 2C–E). The experiment
performed with PIII verifies that the three fragments
observed with probes F, PI and PII originate from the 39
end of the probes. Also, a protected fragment corresponding
to a transcript beginning at nt 480 was observed using
PII (Fig. 2D). In the experiment performed with PI, a
protected fragment with the length of approximately
174 bp was observed. This fragment was not identified in
the experiments using PII, but could correspond to the
unidentified fragment observed using probe F (Fig. 2B). The
experiments were repeated several times and the same
fragments appear using the two different probes (F and
PI); therefore, it must be a specific, protected fragment.
The origin of this fragment is still unknown. In conclusion,
the four different probes reproducibly protect fragments
corresponding to the P97 and P480 mRNAs described
previously, but also fragments corresponding to 59 ends
around nt 441 and 430. Therefore, these experiments
support the presence of transcripts initiating between nt
430 and 441.
Promoter activity of luciferase reporter
constructs
To examine the promoter activity associated with the novel
transcription start sites, different HPV-16 fragments were
amplified by PCR and inserted into the pGL3-Enhancer
vector, which contains the SV40 enhancer and the luciferase
reporter gene. The SV40 enhancer was used as a substitute
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M. W. Rosenstierne and others
Fig. 1. (A) PCR amplification of cDNA using the special cap primer (rXhoICap) and the HPV-16-specific primer (770bHindIII).
The transcripts expected are summarized. (B) PCR products were visualized on a 1?2 % agarose gel. The outer lanes show
the pBR322-HinfI marker. Two different PCR reactions are shown in duplicate. The different products corresponding to
different transcripts are shown. (C) PCR fragments were purified and inserted into the pGL3-Basic vector. Different clones
were sequenced using a vector-specific primer. The first sequence shows a transcript beginning at nt 97 (the main promoter
P97). The arrow points to the first nucleotide in the HPV-16 sequence. Sequences of the different transcription start
sites identified in the E6 ORF of HPV-16 are shown, and the dotted lines separate the DNA sequences originating
from the pGL3-Basic vector, the XhoI restriction site, the CapFinder sequence and the HPV-16 sequence.
for the HPV enhancer (LCR), which, in the natural context
of the full-length HPV genome, will influence the activity
of different HPV promoters. Constructs were designed to
include the novel transcription start sites at nt 430, 441
and 446 either alone (nt 272–448) or together with the
transcription start site identified previously at nt 480 (nt
272–496) (Fig. 3A). The different pGL3-Enhancer constructs were transiently transfected into HPV-16-positive
SiHa cells, HPV-18-positive HeLa cells and HPV-negative
C33A cells. Luciferase activity was measured 48 h after
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transfection (Fig. 3B). Each construct was tested twice in
triplicates with two different batches of DNA, resulting in
at least 12 measurements for each construct. The 272–448E
construct had a significantly higher luciferase activity than
the empty pGL3-Enhancer vector in all three cell lines tested,
with the highest activity in the SiHa cell line (8?5-fold
increase). The 272–496E construct had lower activity than
the 272–448E construct in SiHa and HeLa cells but in the
C33A cell line both fragments showed similar activity (2fold increase). A short construct (448–496E) that contained
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HPV-16 and transcription
only the transcription start site at nt 480 had no activity.
These results demonstrate that the region at nt 272–
448 is needed to activate transcription from nt 480. To
identify the essential regions responsible for promoter
activity, upstream and downstream deletions were made
(Fig. 3A). Deletion of the region containing the transcription start sites reduced luciferase activity to that of the empty
pGL3-Enhancer vector. Deletions of the upstream region
also showed a luciferase activity corresponding to that of the
empty vector, except for the 332–496E construct in the SiHa
cell line, which showed a 1?5-fold increase in activity; this,
however, is a 5-fold reduction compared to that seen with
construct 272–496E (Fig. 3B). These results indicate that the
region from nt 272 to 332 is essential for transcription
initiation from the two clusters of transcription start sites
around nt 441 and 480.
proteins bind specifically to this region. No specific binding
of proteins from the SiHa nuclear cell extract to the
intermediate region from nt 332 to 388 was observed. In
contrast, the HeLa nuclear cell extract (Fig. 4D, lane 4)
showed binding to the probe specific for nt 332–388, which
could be competed by the nt 332–354 competitor fragment.
However, a 50-fold excess of competitor could not remove
the shifted band completely and the specificity of this
binding therefore remains unclear. The region from nt 388
to 448 showed four shifted bands, with both the SiHa and
the HeLa nuclear cell extracts (Fig. 4F, lane 7). The binding
region was within nt 388–411. These results demonstrate that proteins from SiHa and HeLa nuclear cell
extracts bind specifically to the region upstream of the nt 441
transcription start sites.
To verify that the promoter activity observed was not due to
unspecific initiation of transcription caused by the strong
SV40 enhancer, different fragments were also inserted into
the pGL3-Basic vector, which lacks the SV40 enhancer. The
pGL3-Basic vector was modified further to reduce background activity, as described by Braunstein et al. (1999). In
these experiments, the HPV-16 fragments contained only
the transcription start sites around nt 441 (Fig. 3C). The
same upstream and downstream deletions as mentioned
above were constructed. Transient transfections were performed using the SiHa cell line because this cell line showed
the highest luciferase activity (Fig. 3B). Construct m272–
448B showed considerably higher activity (103-fold) than
the empty pGL3-Basic vector. Deletions of the region
containing the transcription start sites all reduced luciferase
activity to that of the empty vector. Again, an approximately
5-fold reduction was seen for construct m332–448B. In
conclusion, these results confirm that promoter activity is
present within the region of nt 272–448 and that this activity
is not an artefact introduced by the strong SV40 enhancer.
Furthermore, the experiments confirmed that the region at
nt 272–332 is essential for activation of transcription from
the novel start sites around nt 441.
Transcription is independent of a TATA-box-like
sequence
Binding of proteins to the region at nt 272–448
To investigate binding of transcription factors to the region
of nt 272–448, EMSA analysis was performed with SiHa
and HeLa nuclear cell extracts. DNA probes spanning the
regions at nt 272–332, 332–388 and 388–448 were 59
end-labelled with [a-32P]ATP. All three probes were shifted
by both nuclear extracts (Fig. 4B, D and F, lanes N).
Competition experiments were performed with full-length,
non-labelled probes (data not shown) or smaller (approximately 23 bp) overlapping DNA probes, as illustrated in
Fig. 4(A, C and E). The binding of HeLa nuclear cell extract
to the probe specific for nt 272–332 was competed by the
nt 291–314 competitor fragment (Fig. 4B, lane 2). This
indicates that a protein binds specifically in this region. The
experiments performed with SiHa nuclear cell extract
showed competition of two of the shifted bands within
the same region (Fig. 4B, lane 2), again indicating that
http://vir.sgmjournals.org
Two TATA-box-like elements are present upstream of
the transcription start sites around nt 441. One element is
positioned at nt 401–407 with the sequence 59-TTAATTA-39
(Fig. 5A, mut I), which is in the protein-binding region
of nt 388–411 identified by EMSA (Fig. 4F, lane 7). The
second element is situated immediately adjacent to the
protein-binding region at nt 412–417 with the sequence
59-TATTAA-39 (Fig. 5A, mut II). Point mutations were
introduced into both elements to investigate influences on
the activity of the start sites around nt 441. Fragments
amplified by PCR were cloned into the pGL3-Enhancer
vector, sequenced and transfected into the C33A, HeLa and
SiHa cell lines. Luciferase activity was measured subsequently. No significant difference in the promoter activity
between the wild-type and mutant constructs was observed
(Fig. 5B). These results show that transcription from the
cluster of transcription start sites around nt 441 functions
independently of the TATA-box-like element.
DISCUSSION
The data presented here identify a novel cluster of transcription start sites around nt 441 in the E6 ORF of the HPV16 genome. Initiation sites were mapped with a 59RACE
PCR method. In addition to the new initiation sites, we
mapped transcripts from P97 to initiate at nt 97 and 95. We
also found a cluster of transcription start sites present at
around nt 480, which have been described previously as the
promoter P480 (Grassmann et al., 1996; Braunstein et al.,
1999). To verify the presence of transcription start sites
around nt 441, we performed RNase protection assays with
four different probes. The experiment performed with PIII
was designed to verify that all protected fragments observed
using the probes F, PI and PII originated from the 39 end
of the probes. Probe F, PI and PII repeatedly identified
protected fragments corresponding to transcripts from P97,
including the different splice patterns. In addition, fragments that correspond to transcripts initiated from P480, nt
441 and 430 were identified with the same three probes and
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M. W. Rosenstierne and others
thus show that HPV-16 transcripts initiating around nt 441
are present in the CaSki cell line.
Polymerase II promoters with several start sites are usually
not dependent on a TATA-box element (Ince & Scotto,
1995). Accordingly, we find that point mutations made in
the two TATA-box-like elements present upstream of the
multiple transcription start sites did not influence promoter
activity. The data, therefore, support the conclusion that
transcription initiation from the several novel transcription
start sites is independent of a TATA-box element. Comparison of the sequence surrounding the transcription start
sites did not reveal any core elements or initiator sequences
(for reviews, see Butler & Kadonaga, 2002; and Ince &
Scotto, 1995). Promoters with multiple transcription start
sites and no recognizable core elements have also been
identified in other regions of the genomes from a number of
different HPV types. They include the late differentiationdependent promoters (DiLorenzo & Steinberg, 1995;
Grassmann et al., 1996; Karlen et al., 1996; Klumpp &
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Laimins, 1999; Nasseri et al., 1987; Ozbun & Meyers, 1997,
1998; del Mar Peña & Laimins, 2001; Tomita et al., 1996).
Some of these transcription start sites are scattered over
relatively large regions of the genome; for example, P670
(HPV-16) has transcription start sites spanning approximately 100 nt from nt 667 to 766 (Grassmann et al., 1996).
In HPV-31b, 30 transcription start sites have been mapped
to a region spanning approximately 200 nt in the E7 ORF
(del Mar Peña & Laimins, 2001). Considering these findings,
it seems likely that the transcription start sites clustered
around nt 441 and P480 are part of a large cluster of
transcription initiation sites and are controlled by the same
sequence elements. Our finding supports this view, since the
promoter activity of the fragment at nt 448–496 does not
show any promoter activity unless the upstream region (nt
272–448) is included. Furthermore, our finding suggests
that the upstream region at nt 272–332 is required for
transcription activity. Possibly, this region recruits the
general transcription machinery or parts thereof and, in
the absence of consensus core promoter signals, initiates
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HPV-16 and transcription
Fig. 2. RNase protection assay. (A) Locations of the different promoters of the HPV-16 genome. The HPV-16-specific
probes F (nt 53–632), PI (nt 277–526), PII (nt 409–600) and PIII (nt 277–431) and their expected fragments originating from
the different splice patterns of transcripts and initiation sites are shown. Crossed boxes indicate part of the linker region from
the linearized vector, 30 nt from the pAlter-1 vector on probe F and 60 nt from the Bluescript IISK+ vector on probes PI, PII
and PIII. (B) RNase protection gel using probe F. Reactions using 5–10 mg CaSki RNA are shown in lanes F. Fragments
corresponding to the different specific HPV transcripts are shown. RNase protection gel using PI (C), PII (D) and PIII (E).
Fragments corresponding to the different specific HPV transcripts are shown. Lanes: M, RNA marker (bp); U, 10 % of the
probe used in the experiment without CaSki RNA; C, control for the specificity of the probe using 20 mg yeast RNA.
transcription from several positions within the region of
nt 388–496.
Many of the late promoters with multiple transcription start
sites that have been identified in the different HPV types are
dependent on differentiation. In HPV-31, only some of the
multiple transcription start sites clustered around the P742
promoter are active in non-differentiated cells, but upon
differentiation a number of these transcription start sites are
activated (Ozbun & Meyers, 1998; del Mar Peña & Laimins,
2001). It has also been shown that the E7 promoter of HPV6 is upregulated in differentiated cells (Ai et al., 1999). One
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possible mechanism for this upregulation is differentiationdependent downregulation of the repressor protein (CDP/
cut). This protein is present in non-differentiated cells
and binds to AT-rich MAR (nuclear matrix attachment)
elements in the promoter region. A similar MAR element is
present in the E6 ORF of HPV-16 (Tan et al., 1998) and this
region also binds the CDP/cut repressor (Stünkel et al.,
2000). Our data confirm protein binding to this region.
By EMSA analysis, we found four band shifts within the
region of nt 388–411 that could be specifically competed.
Identification of the binding proteins is currently under
investigation. A strong candidate is the CDP/cut repressor,
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M. W. Rosenstierne and others
Fig. 3. Luciferase reporter assay. (A) The two clusters of transcription start sites around nt 441 and 480 are shown together
with the different DNA fragments inserted into the pGL3-Enhancer vector. The different coordinates are given. (B) All
fragments inserted into the pGL3-Enhancer vector are indicated by the suffix E. Luciferase activity is given as a percentage of
the empty pGL3-Enhancer vector. Two different batches of DNA were used for each construct and tested twice in triplicates
with a total of 12 measurements. Bars indicate SD. (C) The different DNA fragments inserted into the pGL3-Basic vector are
shown together with the coordinates. (D) The pGL3-Basic constructs were all modified to reduce background activity and are
therefore given the prefix m in addition to the suffix B. Luciferase activity is given as a percentage of the empty modified pGL3Basic vector. Two different batches of DNA were used for each construct and were tested twice in triplicates with a total of
12 measurements. Bars indicate SD.
since Stünkel et al. (2000) have identified a strong binding of
CDP/cut to an oligonucleotide spanning the region of nt
374–403. It would be interesting to study the influence of
2916
cell differentiation on the transcription activity of the cluster
of transcription start sites around nt 441 and we plan to
address this in future studies.
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Journal of General Virology 84
HPV-16 and transcription
Fig. 4. Binding of protein to the promoter region. (A, C and E) The different probes and competitors with coordinates are
shown. Numbers 1–9 designate the small competitors. (B, D and F) EMSA gels using HeLa and SiHa nuclear cell extracts.
Reactions with nuclear extract but without competitor are seen in lanes N. The different competitors added in three increasing
concentrations are shown for each gel. Arrows point towards the specific fragments that respond to competition.
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M. W. Rosenstierne and others
Fig. 5. Site-directed mutagenesis. (A) The sequence of nt 388–448 containing the cluster of transcription start sites around
nt 441 is shown. The protein-binding region is marked with a grey background. TATA-box-like elements are shown in bold and
mutated nucleotides are underlined. Adenine was changed to cytosine and thymidine was changed to guanine. (B) Luciferase
activity in the three cell lines is given as a percentage of the empty pGL3-Enhancer vector. Two different batches of DNA
were used for each construct and tested twice in triplicates with a total of 12 measurements. Bars indicated SD.
Previously, Stünkel et al. (2000) have shown that the E6
MAR element of HPV-16 is a strong cis-responsive element
when situated downstream of P97 in a reporter construct.
In transient transfection studies, the E6 MAR element
repressed the promoter activity of P97. However, in stable
transfections with the constructs integrated into the host
genome, the promoter activity of P97 was enhanced.
Deletion constructs showed that the region at nt 246–356
had to be included in the constructs in order to activate P97
when integrated in the host genome. We found that the
region at nt 272–332 is essential for the promoter activity of
nt 441. Our results, together with the data presented by
Stünkel et al. (2000), indicate therefore that the region at
nt 272–332 is a strong cis-responsive element, which, in the
natural context of the genome, might work on both the main
promoter P97 and the cluster of transcription start sites
around nt 441.
In summary, our data show that there is a new cluster of
transcription start sites around nt 441 in the E6 ORF of
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HPV-16. The transcription start site clusters around nt 441
and 480 and the P542 promoter identified previously all
produce messengers with potential to express E7. Although
the P441, P480 and P542 messengers are expressed at low
levels compared with P97 messengers, they may contribute
considerably to the overall expression of E7 through more
efficient translation. Therefore, the P441, P480 and P542
promoters could be potentially important for malignant
progression of HPV-16-infected cells.
ACKNOWLEDGEMENTS
We acknowledge Harald zur Hausen (DKFZ, Heidelberg) for the
pBR322 plasmid containing the HPV-16 genome. We thank Med
Jørgen Olsen (Biochemistry Department C, Panum Institute) for
advice and technical guidance on the preparation of SiHa nuclear cell
extracts and EMSA analysis. This work was supported by grants from
the Danish Cancer Institute, the Danish Medical Research Council,
the Biotechnology Foundation, the Novo Nordisk Foundation, the
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Journal of General Virology 84
HPV-16 and transcription
Glud Foundation, the Arvid Nilsson Foundation, the Wedell
Wedellsborg Foundation, the Klestrup Foundation, the Direktør E.
Danielsen Foundation and the Dagmar Marshall Foundation.
Nishimura, A., Ono, T., Ishimoto, A., Dowhanick, J. J., Frizzell, M. A.,
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cervical cancer cell growth inhibition. J Virol 74, 3752–3760.
O’Connor, M. J., Chan, S. Y. & Bernard, H.-U. (1995). Transcription
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