Download The bovine papillomavirus type 4 long control region contains an

Journal
of General Virology (1999), 80, 23–27. Printed in Great Britain
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SHORT COMMUNICATION
The bovine papillomavirus type 4 long control region contains
an epithelial specific enhancer
Iain M. Morgan, G. Joan Grindlay and M. Saveria Campo
Beatson Institute for Cancer Research, CRC Beatson Laboratories, Garscube Estate, Glasgow G61 1BD, UK
Bovine papillomavirus type 4 (BPV-4) is a mucosal
epitheliotropic virus that is a causative agent in
alimentary carcinoma of cattle. The long control
region (LCR) of this virus controls expression of the
transforming proteins, E8 and E7. Deletion mutants
of the LCR were prepared and assayed for their
ability to activate transcription from the LCR promoter in primary bovine palate keratinocytes (the
natural target cell for BPV-4) and fibroblasts. The
LCR was at least an order of magnitude more active
in keratinocytes than in fibroblasts. An epithelial
specific enhancer was identified that activated
transcription from the SV40 promoter to levels
identical to the full-length LCR. One of the active
sites in the enhancer is 100 % conserved in the LCR
of human papillomavirus type 16. The results
demonstrate that the BPV-4 LCR has an epithelial
specific enhancer, which offers the opportunity to
study epithelial specific transcriptional regulation
of papillomavirus promoters.
Human papillomaviruses (HPV) are causative agents of
squamous cell carcinomas, with at least 90 % of cervical
carcinomas containing HPV sequences (zur Hausen, 1991). The
mucosal epitheliotropic viruses HPV-16 and -18 are the most
common viruses associated with cervical carcinoma and one of
the mechanisms restricting these viruses to epithelial cell types
involves transcriptional control. The transforming genes of
these viruses, E6 and E7, are controlled by the long control
region (LCR), which can be divided into three regions, the
promoter region, the enhancer region and an area upstream of
the enhancer that is apparently not involved in transcriptional
regulation (Desaintes & Demeret, 1996). The enhancer region
of these viruses is epithelial specific, as it fails to activate
transcription from heterologous promoters in non-epithelial
cell types (Gloss et al., 1987). A multiplicity of factors binds to
Author for correspondence : Iain Morgan.
Fax j44 141 942 6521. e-mail i.morgan!beatson.gla.ac.uk
0001-5849 # 1999 SGM
these epithelial specific enhancers, including AP-1 (Offord &
Beard, 1990 ; Thierry et al., 1992 ; Chong et al., 1991), Oct-1
(Hoppe-Seyler et al., 1991 ; Morris et al., 1993), NF1 (Apt et al.,
1993, 1994 ; O’Connor & Bernard, 1995), PEF-1 (Cuthill et al.,
1993 ; Sibbet et al., 1995), Sp1 (Apt et al., 1996) and YY1
(O’Connor et al., 1996 ; Dong et al., 1994), although no one
factor has been identified that determines the epithelial specific
nature of these enhancer elements.
Bovine papillomavirus type 4 (BPV-4) is a mucosal
epitheliotropic papillomavirus and is a causative agent of
alimentary canal carcinoma in cattle (Campo et al., 1994). Like
HPV-16 and -18, the LCR of BPV-4 controls the expression of
the transforming genes of the virus, E8 and E7. Although the
distribution of E2 DNA-binding sites within the LCR is highly
conserved between mucosal epitheliotropic papillomaviruses
from different species, the overall sequence similarity is quite
low (Morgan et al., 1998). This suggests that BPV-4 uses a
different combination of cellular factors from HPV-16 and -18
to control transcription from the LCR promoter. Previous
studies have shown that the LCR of BPV-4 contains negative
and positive control elements that regulate transcription from
the TK promoter (Jackson & Campo, 1991). However, it is not
known whether the transcriptional control elements of this
viral LCR are epithelial specific.
To investigate the regions of the LCR involved in
regulating transcription from the BPV-4 LCR promoter,
various deletion mutations were introduced into the LCR
cloned upstream of the luciferase gene in the vector pGL3
(Promega). These constructs were used to identify elements in
the LCR that contribute towards transcriptional activity in
primary bovine palate keratinocytes (PalKs) and fibroblasts
(PalFs), by transfecting these cells and determining the
luciferase activity. The transfection and luciferase assay
protocols for these cell types have been described previously
(Morgan et al., 1998) and the results are shown in Fig. 1. When
compared with the minimal promoter region (represented by
∆184), there was an approximately 400-fold increase in
transcription with the full-length LCR (∆6710) in PalKs.
Deletion from nucleotide 7131 to 7180 resulted in a 3-fold
drop in activation from the LCR promoter, while deletion from
nucleotide 20 to 44 resulted in a 10-fold drop in activation. We
have named these two regions Site1 and Site2, and they
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I. M. Morgan, G. J. Grindlay and M. S. Campo
(a)
Fig. 2. The BPV-4 LCR contains an epithelial specific enhancer. The two
main sites contributing towards transcriptional activation (Fig. 1 b) were
amplified by PCR and cloned upstream of the SV40 promoter in the vector
pGL3PRO (see text for details). The resulting constructs were tested for
their ability to activate transcription from the SV40 promoter in PalKs and
PalFs. Ten µg of each plasmid was used in each experiment and the
results are expressed as fold activation over pGL3PRO. The results
represent at least three sets of experiments carried out in duplicate. SV,
SV40 promoter alone ; LCR, full-length BPV-4 LCR cloned upstream of the
SV40 promoter ; ENj, the area of the BPV-4 LCR from nucleotide 7131
to 44 (a total of 178 bp) cloned in the forward orientation upstream of
the SV40 promoter ; EN-, the area of the BPV-4 LCR from nucleotide
7131 to 44 (a total of 178 bp) cloned in the reverse orientation upstream
of the SV40 promoter.
(b)
(c)
Fig. 1. The BPV-4 LCR has enhanced transcriptional activity in epithelial
cells. (a) The deletion mutations shown of the BPV-4 LCR were cloned
upstream of the luciferase gene in the vector pGL3. These vectors were
then transfected into (b) PalKs or (c) PalFs and the resulting luciferase
activity was determined. Ten µg of each plasmid was used in each
experiment and the results are expressed as fold activation over the ∆184
vector. Triangles represent binding sites for the papillomavirus
transcription factor E2. The results represent at least three sets of
experiments carried out in duplicate.
account for 99 % of the transcriptional activity of the LCR in
PalKs. In PalFs, the level of transcriptional activation was
approximately 30-fold less than that seen in PalKs, although
the overall pattern of transcriptional activity of the deletion
mutants was similar.
In the HPV-16 and -18 LCRs, there are regions that
function as epithelial specific enhancers when located upstream
of heterologous promoters (Gloss et al., 1987). The results
presented in Fig. 1 suggest that the BPV-4 LCR contains an
enhancer element between nucleotides 7131 and 44. To test
this possibility, a DNA fragment from 7131 to 44 (178 bp) was
PCR-amplified with Pfu1 (Stratagene) and cloned upstream of
the SV40 promoter in the vector pGL3PRO (Promega), in both
CE
the sense and anti-sense orientations, as a KpnI–HindIII
fragment. The resulting constructs were sequenced and then
assayed for transcriptional activity in PalKs and PalFs, and the
results are shown in Fig. 2. In the forward or reverse
orientation, this fragment enhanced transcription from the
SV40 promoter in PalKs 6-fold, which was an identical
enhancement to that seen with the full-length LCR. The
relative level of activation from the SV40 promoter was lower
than that obtained with the BPV-4 promoter (Fig. 1), as the
background activity of the SV40 promoter was much higher in
both keratinocytes and fibroblasts. The enhanced transcription
was driven exclusively from the SV40 promoter, as the BPV4 enhancer described here contains no transcriptional start sites
(Jackson & Campo, 1995). These results indicate that nucleotides 7131 to 44 contain the enhancer region of the BPV-4
LCR. From Fig. 2 it is also clear that the enhancer element of
BPV-4 does not enhance transcriptional activation from the
SV40 promoter in fibroblasts. This result is in agreement with
studies of other epitheliotropic papillomavirus enhancers that
operate in an epithelial cell type-specific manner (Desaintes &
Demeret, 1996).
To map the sequences that are important for the BPV-4
LCR enhancer function in more detail, further small deletions
were introduced into Site1 and Site2. This was done by
amplifying the sequences from the LCR, down to the promoter,
by PCR with Pfu1 and cloning them in front of the luciferase
gene. All of the constructs were sequenced and the transcriptional activities of the mutants were determined in PalKs.
The results of these experiments are shown in Fig. 3 (a, b),
together with the sequences. For Site1, there are two regions
that contributed to the drop in activity observed between
∆7131 and ∆7176 (which showed the same level of activity as
∆7180 shown in Fig. 1), nucleotides 7131 to 7142 and 7150 to
7158. Overall, there was a 2n6-fold drop in activity with these
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Epithelial specific BPV-4 transcription
(a)
(b)
(c)
Fig. 3. Further mapping of the sites contributing to BPV-4 LCR transcriptional activity in PalKs. Site1 (a) and Site2 (b) were
further analysed by deletion of the sites by PCR, as detailed in the text. All of the inserts were cloned into pGL3 and 10 µg
aliquots of the resulting constructs were transfected into PalK and their transcriptional activity was determined. The results are
expressed as a percentage of the activity of constructs containing the complete Site1 or Site2. In (b), the 9 bp sequence in
Site2 that is 100 % conserved with the LCR of the HPV-16 LCR is underlined. (c) Mutations were introduced into the 9 bp
sequence in Site2 and are shown in lower case letters in the underlined area of Site2. The results are expressed as a
percentage of the activity obtained with ∆15. The results represent at least three sets of experiments carried out in duplicate.
two deletions, compared with a 3n1-fold drop observed in Fig.
1 with the same region deleted. For Site2, there were also
regions that when deleted led to a loss of transcriptional
activity : nucleotides 15 to 23 and 23 to 29, representing 14
nucleotides overall. The activity of ∆29 in Fig. 3 was the same
as that for ∆44 in Fig. 1. There is a sequence in the LCR of HPV16 that is identical to a 9 bp sequence in Site2, TGTGTAAAG,
which is highlighted in Fig. 3 (b). This sequence is located at
position 7800 in HPV-16, which lies outside the enhancer
region, although there is a cellular protein that binds to this
sequence (Nakshatri et al., 1990 ; Gloss et al., 1989). The
contribution made by this sequence to the transcriptional
control of the HPV-16 LCR is not known, as the YY1 sites that
repress the HPV-16 promoter make the study of this sequence
difficult in the context of the HPV-16 promoter. To determine
whether this sequence is responsible for the activity of Site2,
mutations were introduced into the 9 bp region and the
transcriptional activity of the mutants was assayed. The
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I. M. Morgan, G. J. Grindlay and M. S. Campo
mutations and the results from these experiments are shown in
Fig. 3 (c), and it is clear that the 9 bp sequence is essential for
transcriptional activity from Site2. The 9 bp sequence bears no
close similarity to any known transcription factor-binding site.
The results demonstrate that the BPV-4 LCR has an
organization typical of other mucosal epitheliotropic papillomavirus LCRs. There is a promoter region, an enhancer region and
an area upstream that does not appear to contribute towards
transcriptional regulation by cellular factors. The upstream
binding site for the papillomavirus transcription factor E2, BS4,
lies outside the enhancer region, as it does in the HPV-16 and
-18 LCRs. The distribution of the E2 DNA-binding sites in
mucosal epitheliotropic papillomavirus LCRs is highly conserved, indicating that the mechanism that E2 uses to regulate
transcription is conserved (Morgan et al., 1998). However, the
cellular factors that BPV-4 uses to achieve epithelial specific
transcriptional regulation may be distinct from those used by
the HPVs. For example, there is no Sp1 site in the BPV-4 LCR
upstream of the E2 sites proximal to the TATA box, as there
is in HPV LCRs (Apt et al., 1996). There are also no obvious
AP-1 sites in the BPV-4 LCR, which is again distinct from the
HPV LCRs, where these sites have a positive effect on
transcription from adjacent papillomavirus promoters (Offord
& Beard, 1990 ; Thierry et al., 1992 ; Chong et al., 1991).
However, there are several transcription factors that are
implicated in transcriptional control from the HPV and BPV-4
LCRs. As mentioned above, the transcription factor family
C\EBP has been implicated as both a positive and negative
regulator of transcription from HPV and BPV-4 LCRs
(Bauknecht et al., 1996 ; Bauknecht & Shi, 1998 ; McCaffery &
Jackson, 1994 ; Wang et al., 1996). Also, there are several
putative binding sites for the NF1 transcription factor family in
the BPV-4 LCR (Jackson & Campo, 1991). It has been well
documented that this family is involved in regulating transcription from HPV LCRs (Apt et al., 1993, 1994 ; O’Connor &
Bernard, 1995).
Exactly what combination of transcription factors is
responsible for mediating the epithelial specific transcriptional
regulation of papillomavirus LCRs remains to be established. It
seems likely that a combination of ubiquitous factors, or
specific members of transcription factor families, act in concert
to establish a pattern of epithelial specific transcriptional
regulation. Here, we have demonstrated that the BPV-4 LCR
has an epithelial specific enhancer that has two main regions
contributing towards this enhancer activity. Footprinting
studies have demonstrated that cellular factors bind to these
two regions (Jackson & Campo, 1991). One of the sites, Site2,
has a corresponding sequence in the LCR promoter region of
HPV-16. Identification of the proteins involved and the
contribution of the individual elements to epithelial specificity
is under way. The BPV-4 LCR presents a further opportunity
to study the mechanisms that mucosal epitheliotropic
papillomaviruses use to achieve epithelial specific transcriptional regulation.
CG
We thank Dr John Wyke for critical reading of the manuscript. This
work was supported by the Cancer Research Campaign.
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Received 27 July 1998 ; Accepted 28 September 1998
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