Institute of Molecular Pathology, The Protein Laboratory, University of Copenhagen, Panum Institute, Bldg 6.2, Blegdamsvej 3C, DK.2200 Copenhagen N., Denmark
Correspondence
Bodil Norrild
bono{at}biobase.dk
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ABSTRACT |
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Published ahead of print on 27 August 2003 as DOI 10.1099/vir.0.19332-0.
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INTRODUCTION |
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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 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 272448 and we show that several nuclear proteins from the SiHa and HeLa cell lines bind to two terminal sites at nt 291314 and 388411 of this region. Furthermore, we find that the fragments spanning nt 272332 and 388448 are important for the transcription activity of the region at nt 272448. We also show that transcription activity is independent of the presence of a TATA-box-like element.
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METHODS |
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5'RACE PCR and cloning.
The 5'RACE 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 µg µl-1) were added to 5x 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, 5'-aagcagtggtatcaacctcgagtacgc(ggg)r-3'). 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 °C for 4 min, followed by five cycles of 94 °C for 30 s, 66 °C for 1 min and 72 °C for 1 min, five cycles of 94 °C for 30 s, 62 °C for 1 min and 72 °C for 1 min, and 20 cycles of 94 °C for 30 s, 58 °C for 1 min and 72 °C for 1 min. The primers used were an HPV-16-specific primer (770bHindIII, 5'-catggacaagctttgtacgcacaaccgaagcgtagagtcacac-3'), containing an HindIII restriction site, and the cap primer (XhoICap, 5'-aagcagtggtatcaacctcgagtacgcggg-3'), containing an XhoI site. cDNA was then visualized on a 1·2 % agarose gel. A 2 µl sample of the PCR mixture (100 µl) was used in a new PCR protocol using the programme: one cycle of 94 °C for 4 min, followed by 20 cycles of 94 °C for 30 s, 58 °C for 1 min and 72 °C 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 272291, 332352, 388409, 448463, 332314, 388370, 448427 and 496478. 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 272448 fragment was performed using primers containing two point mutations (5'-gcatggacgctagccttttcttcaggacacagtggcttttgacagttaatacacctacgtaacaaatca-3' and 5'-gcatggacgctagccttttcttcaggacacagtggcttttgacagtgaagacacctaatt-3'; mutations are shown in bold). The PCR product was amplified using Taq polymerase (Amersham Pharmacia Biotech) to avoid 3'
5' 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 53632 fragment was amplified by PCR (5'-atggacggatccgaaaccggttagtataaaagcagac-3' and 5'-gtggacgaattccagtagagatcagttgtctc-3') 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 277526, 409600 and 277431 were amplified by PCR using the following primers: PI, 5'-gcatggacctcgagtccatatgctgtatgtgataaatg-3' and 5'-gcatggacggtacctctgcaacaagacatacatcgaccg-3'; PII, 5'-gcatggacctcgaggtgtattaactgtcaaaagccactg-3' and 5'-gcatggacggtacctaacatatattcatgcaatgtaggtg-3'; PIII, 5'-gcatggacctcgagtccatatgctgtatgtgataaatg-3' and 5'-cgttaggtaccgtggcttttgacagttaatacacc-3'. 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 [-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 µg CaSki RNA. Probes PII (87 000 c.p.m.) and PI and PIII (80 000 c.p.m.) were mixed with 10 µg 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 spanning nt 272448, 272332, 332388 and 388448 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 5' overhang. Probes were purified using 1 % agarose gels, according to the manufacturer's instructions (Qiagen). Probes were labelled with [-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 µg HeLaScribe nuclear cell extract (Promega) or 15 µg SiHa nuclear cell extract, purified according to the procedure described by Dignam et al. (1983)
, were used together with 0·1 pmol probe µl-1. 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.
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RESULTS |
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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 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 272448) or together with the transcription start site identified previously at nt 480 (nt 272496) (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 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 272448E 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 272496E construct had lower activity than the 272448E construct in SiHa and HeLa cells but in the C33A cell line both fragments showed similar activity (2-fold increase). A short construct (448496E) that contained only the transcription start site at nt 480 had no activity. These results demonstrate that the region at nt 272448 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 332496E 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 272496E (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.
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Binding of proteins to the region at nt 272448
To investigate binding of transcription factors to the region of nt 272448, EMSA analysis was performed with SiHa and HeLa nuclear cell extracts. DNA probes spanning the regions at nt 272332, 332388 and 388448 were 5' end-labelled with [-32P]ATP. All three probes were shifted by both nuclear extracts (Fig. 4
B, 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 272332 was competed by the nt 291314 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 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 332388, which could be competed by the nt 332354 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 388411. 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.
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DISCUSSION |
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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 differentiation-dependent promoters (DiLorenzo & Steinberg, 1995
; Grassmann et al., 1996
; Karlen et al., 1996
; Klumpp & 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 448496 does not show any promoter activity unless the upstream region (nt 272448) is included. Furthermore, our finding suggests that the upstream region at nt 272332 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 transcription from several positions within the region of nt 388496.
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 HPV-6 is upregulated in differentiated cells (Ai et al., 1999
). One possible mechanism for this upregulation is differentiation-dependent 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 388411 that could be specifically competed. Identification of the binding proteins is currently under investigation. A strong candidate is the CDP/cut repressor, since Stünkel et al. (2000)
have identified a strong binding of CDP/cut to an oligonucleotide spanning the region of nt 374403. It would be interesting to study the influence of 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.
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 246356 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 272332 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 272332 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 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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 6 May 2003;
accepted 8 August 2003.