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Identification of cis-regulatory elements in the upstream regulatory region of human papillomavirus type 59
Virus Research 47 (1997) 155 – 166
Identification of cis-regulatory elements in the upstream regulatory region of human papillomavirus type 59 Jaerang Rhoa, Soyoung Leea, Ethel-Michele de Villiersb, Joonho Choea,* a
Department of Biological Sciences, Korea Ad6anced Institute of Science and Technology, Taejon 305 -701, South Korea b Di6ision Tumour6irus-Characterization, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany Received 14 August 1996; revised 16 October 1996; accepted 16 October 1996
Abstract Human papillomavirus type 59 (HPV-59) was cloned from a vulvar intraepithelial neoplasia and the complete nucleotide sequence was determined. This virus is closely related to HPV-18 and -39 (60% homology in nucleotide sequence) and is grouped with the genital HPV types. In the present paper, we demonstrate that the HPV-59 E2 transactivator represses its E6 promoter-mediated transcription. We have also analyzed cis-regulatory elements in the upstream regulatory region (URR) of HPV-59 using chloramphenicol acetyl transferase assays as well as electrophoresis mobility shift assays (EMSA). The results allow for a subdivision of the HPV-59 URR into three regions of activity: distal (nt 7149–7493), central (nt 7493–7742), and proximal (nt 7742 – 7748). In particular, the 250 bp (nt 7493–7742) of the central region plays an important role as a constitutive enhancer element for the maximal transcription of the E6 promoter. Our results suggest that the transcription factors AP1, Oct1, SP1 and unidentified factors bind to the HPV-59 E6 promoter region, whereas NF1, GRE and TFIID fail to bind despite the presence of putative binding sites in the DNA sequence. © 1997 Elsevier Science B.V. Keywords: CAT assay; EMSA; E6 promoter; HPV-59; Upstream regulatory region
1. Introduction Human papillomaviruses (HPVs) are causative agents of a variety of benign and malignant lesions in humans (de Villiers, 1994; zur Hausen, 1991a). Experimental and epidemiological data * Corresponding author. Tel.: + 82 42 8694020; fax: + 82 42 8695260; e-mail: [email protected]
indicate a higher oncogenic potential of certain HPV types associated with lesions such as cervical intraepithelial neoplasia (CIN) or carcinomas. So far, more than 100 HPV types have been identified, including 77 of which the genomes have been characterized (de Villiers, 1994). A large number of these HPV types are closely associated with anogenital carcinomas. In particular, HPV16 and -18 are most frequently present in lesions
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progressing towards malignancy (zur Hausen, 1987) and are grouped as ‘high-risk HPVs’. Other HPV types are similarly associated with malignant progression as such, but are not so frequently detected in populations as in the case of HPV-16 and -18. It is important to characterize these other highly oncogenic HPVs in order to understand whether their low frequency is attributable to the mechanism of their oncogenicity and/or their tissue specificity. Previously we have cloned HPV-59 from a vulvar intraepithelial neoplasia and determined its complete nucleotide sequence (Rho et al., 1994). By comparing the HPV-59 DNA sequence to those of all other HPVs, HPV-59 shares the highest nucleotide homology with HPV-18 (71%), HPV-45 (70%) and HPV-39 (69%), all three belonging to the high-risk group (de Villiers, 1994). HPV-59 DNA was detected in CIN I or CIN II lesions in a large scale studies of analyzed clinical samples (Matsukura and Sugase, 1995; de Roda Husman et al., 1994). HPV-59 is present in populations throughout the world, but is nevertheless only detectable in a small percentage of genital lesions. It has, in addition, been associated with lesions on muco-epithelial junctions (Roy-Burman and de Villiers, unpublished data) as well as in oral lesions. Due to its apparent different biological behaviour from already characterized HPVs, it was of interest to us to analyze the regulation of gene expression in HPV-59. The persistent expression of specific HPV viral transcripts and their proteins has been shown to be necessary for the maintenance of malignant cell growth (von Knebel Doeberitz et al., 1992). In particular, gene products of the E6, E7 open reading frames (ORF) in HPV and E5 ORF in bovine papillomavirus type 1 (BPV1) have been identified as having transforming functions (zur Hausen, 1991b). Transcripts coding for E6, E7 and E5 are initiated from the upstream regulatory region (URR) of the HPV genome, just upstream of the E6 ORF (Romanczuk et al., 1990). The initiation of transcription from the URR is controlled by cellular factors and viral proteins which interact with cellular and viral enhancer elements (Chin et al., 1989; Butz and Hoppe-Seyler, 1993). In addition, HPV URR contains sequences neces-
sary for the regulation of viral DNA replication (Russell and Botchan, 1995) and late gene expression (Choe et al., 1989). The URR therefore plays a key role in regulating the gene functions necessary for the malignant transformation of cells. In this study, we tried to identify transcriptional control elements located in the HPV-59 URR. By sequence alignment, the HPV-59 URR shares the highest homology with the URRs of HPV-18 (62%) and HPV-39 (63%) (Rho et al., 1994). Computer analyses revealed similar characteristics for the HPV-59 URR as previously described for the HPV-16, -18 and -33 (Chan et al., 1989), e.g. a number of consensus sequences for binding sites of transcription factors such as GRE (GGTACANNNTGTTCT), SP1 (GGCGGG) or NF1 (TTGGCA), as well as four E2 binding sites (E2-BS, ACCN6GGT). The positions of the E2BS are highly conserved as in other genital HPVs which have two E2-BSs located close to the TATA box of the E6 promoter. The E2 protein of the HPVs can function either as a transactivator or as a repressor (Choe et al., 1989). It acts as a repressor by preventing the formation of an initiation complex with TFIID or SP1 at the E6 promoter (Tan et al., 1992, 1994; Demeret et al., 1994; Dong et al., 1994). It was of interest to us to test whether these binding sites are utilized by cellular transcription factors. We report here the detailed analysis of the HPV-59 URR using the chloramphenicol acetyltransferase (CAT) assay and gel retardation assay with nuclear extracts. Despite the structural similarity to other HPV URRs, the regulation of early gene expression is modulated differently in HPV59 URR.
2. Materials and methods
2.1. Plasmids The HPV-59 URR DNA was amplified by the polymerase chain reaction (PCR) using the forward primer 59c 7149 (5%-GTTAAGCTTTTGTATGTGTGCATG-3%) at nt 7149 and backward primer 59c 48 (5%-GCGTGCCTTGCTGCAGCCCTTTTCTTTTC-3%) at nt 48 and subcloned
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into the pUC19 vector. This amplified product containing the HPV-59 URR and the E6 promoter was subcloned into the upstream region of the CAT gene of pCAT-Basic reporter vector (Promega) and named p59-wtCAT. To construct a HPV-59 E2 expression plasmid, the HPV-59 E2 ORF (nt 2719 – 3894) was amplified using a forward primer 59 c2719 (5%-GATAAGCTTGGAACGAGGAAGAG-3%) at nt 2719 and a backward primer 59 c3804 (5%-CCTCATCTAGACAATATAGCAGCAG-3%) and subcloned into the pRc/CMV expression vector (Invitrogen). It was named pRc59E2. For transient transfection assays pSV2CAT was used as a positive control (Gorman et al., 1982). The p2CAT DNA contains the BPV1 URR and P2 promoter region attached to a CAT gene (Rho et al., 1993). NDE2, which contains the E2 ORF of BPV1, is a derivative of pC59 E2 (Yang et al., 1985).
2.2. Generation of deletion mutants To construct 5% deletion mutants of HPV-59 URR, p59-wtCAT was used as a template for PCR. The sense strand primers were synthesized as follows: 5%-GAGTAAGCTTCTGTCCCTTTATTG-3% (59 c7307) at nt 7307, 5%-GTTTAAGCTTACCTTTTTGAAC-3% (59c 7493) at nt 7493, 5%-CATAAGCTTGGTGGCGCCCTAATA-3% (59c7650) at nt 7650, 5%-ACTAAGCTTTTTGTCATTGTTAAG-3% (59c7792) at nt 7792, 5%-AATAAGCTTGGGTGTAACCGAAAAC-3% (59c7880) at nt 7880. Amplification was performed with an antisense primer 59c 48 at 94°C, 53°C, and 72°C for 2 min each and for 30 cycles. Each of the five PCR products was cloned into a pCAT-Basic vector and labeled as p59-5d7307, p59-5d7493, p595d7650, p59-In7792, and p59-In7880, respectively. Deletion mutants from the 3% end of HPV-59 URR were constructed as follows. The plasmid p59-wtCAT was linearized by PstI digestion. The linearized DNA was digested with exonuclease BAL31 at 30°C between 1 and 40 min. After digestion, the DNA was incubated with DNA
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polymerase Klenow fragment and deoxynucleoside triphosphates to generate blunt ends before a PstI-linker was added. The deleted DNA was digested with HindIII/PstI and ligated into pUC19. The deletion endpoint of each mutant was determined by dideoxy-sequencing (Sanger et al., 1977). Internal deletion mutants were generated by combining appropriate pairs of 5%- and 3%-deletion mutants. Deletion mutants from the 5%-end were digested with HindIII and blunt-ends were generated by the DNA polymerase Klenow fragment before a SmaI-linker was added. Deletion mutants from the 3%-end were digested with PstI and blunt-ends were generated with T4 DNA polymerase and SmaI-linkers added. Deletion mutants from the 5%-end were digested with SmaI and PstI and the SmaI/PstI fragments were subcloned into the large fragments of the HindIII/SmaI of the 3%-deletion mutants. Six internal deletion mutants were confirmed by PCR amplification.
2.3. Cell culture and transient transfection assay C33A cervical carcinoma cells (obtained from Dr Louise T. Chow) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 4.5 g glucose/l and 100 U/ml penicillin and 100 mg/ml streptomycin in an atmosphere of 5% CO2 at 37°C. One milligram of the HPV-59 URR-E6 promoter plasmids and 2.5 mg of the E2 expression plasmid (pRc59E2) were transfected, unless otherwise stated. The total amount of DNA (10 mg) was kept constant by adding salmon sperm DNA as a carrier. Approximately 5× 105 cells were transfected by calcium-phosphate coprecipitation as previously described (Rho et al., 1993) with the following modifications. The DNA-calcium precipitate was applied to the cells for 6 h followed by a shock with 15% glycerol for 1 min. The cells were then washed three times with phosphate buffered saline (PBS) and cultured in DMEM/FCS. Thirty six hours after the glycerol shock the cell extracts were prepared as follows: cells were frozen (−70°C) and thawed at 37°C three times. CAT assays were performed for 5 h according to the standard protocol (Ausubel et
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al., 1990), with the amount of cell extract adjusted to keep the chloramphenicol conversion rate within linear range (100 – 200 mg). The percent conversion of chloramphenicol to the acetylated form was quantitated by Phosphor-Imager (Molecular Dynamics). Each experiment was repeated at least three times.
2.4. Gel retardation assay and preparation of DNA probes The electrophoresis mobility shift assay (EMSA) was performed essentially according to the manufacturer’s protocol (Promega). The binding reaction was performed using 5 mg of HeLa nuclear extracts in binding buffer (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCl, pH 7.5, and 50 mg/ml poly (dI-dC)). The samples were preincubated with or without a specific competitor DNA (2 pm) at room temperature for 10 min. The consensus oligonucleotides of binding sites for several transcription factors present in the EMSA assay were purchased from Promega to use as competitor DNA. The sequences of these oligonucleotides are: SP1: 5%-ATTCGATCGGGGCGGGGCGAGC-3%, AP1: 5%-CGCTTGATGAGTCAGCCGGAA-3%, GRE: 5%-TCGACTGTACAGGATGTTCTAGC TACT-3%, CTF/NF1: 5%-CCTTTGGCATGCTGCCAATAT-3%,
with HindIII/ApyI and the 126 bp fragment was isolated. The probe 7619–7742 was generated by digestion of p59-In7742/7792 with ApyI/SmaI and the 123 bp fragment was eluted. The probe 7792-48 was constructed by digestion of p595d7792 with HindIII/PstI and the 152 bp fragment was isolated. All 5% ends of these DNA fragments were labelled with 32P-ATP by T4 DNA kinase.
3. Results
3.1. HPV-59 E2 protein represses the transcription of E6 promoter To identify the function of the HPV-59 E2 protein in the transcription of the HPV-59 E6 promoter, we transfected the p59-wtCAT DNA into C33A cells with different amounts of the HPV-59 E2 expressing plasmid (pRc59E2). HPV-59 E2 expression resulted in a repression of the reporter activity to a level of about 30% of the control activity (Fig. 1). When p2CAT DNA, which contains the BPV1 E6 promoter, was cotransfected with pRc59E2, the transcription activity was rather slightly (1.4-fold) activated. BPV1 E2 expression also activated its own E6 promoter-mediated transcription by about 3.4-fold. Therefore, the HPV-59 E2 protein behaves differently from the BPV1 E2 protein in regulating its own E6 promoter.
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P-labeled DNA probe (10 fm) was added to each reaction. The reaction mixture was incubated at room temperature for 20 min, after which gel loading buffer (25 mM Tris – HCl, pH 7.5, 0.02% bromophenol blue, 0.02% xylene cyanol, 4% glycerol) was added. The reaction was analyzed by electrophoresis in a 4% polyacrylamide gel (80:1, acrylamide:bisacrylamide; 2.5% glycerol; 0.5×TBE (45 mM Tris – HCl, pH 8.0, 45 mM boric acid, 1 mM EDTA)). To make DNA probes of HPV-59 URR for EMSA, we constructed several DNA fragments spanning the URR as follows: the probe 7493– 7619 was constructed by digesting p59-5d7493
3.2. Analysis of enhancer elements in the HPV-59 URR To identify enhancer elements in the HPV-59 URR, five deletion mutants from the 5%-end were constructed by PCR amplification as described in Section 2 (Fig. 2A). These plasmids were transfected into C33A cells in the presence or absence of pRc59E2 containing the HPV-59 E2 ORF. As shown on Fig. 3, when the URR was deleted between the 5%-end (nt 7149) and nt 7493 (plasmids p59-5d7307 and p59-5d7493), the transcriptional activity of the URR was not influenced in
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Fig. 1. The functional effect of HPV-59 E2 protein on the HPV-59 E6 and BPV1 E6 promoters. Relative CAT activities of HPV-59 E6 and BPV1 E6 promoters (1 mg of DNA) are shown. The cells were cotransfected with increasing amounts of the HPV-59 E2 or BPV1 E2 expression plasmid, pSV2CAT was transfected as the positive control. Relative CAT activity was defined as the ratio of the percent acetylated chloramphenicol to that obtained with extracts made from cells transfected with p59-wtCAT.
comparison to the full-length URR (p59-wtCAT). However, when the URR was deleted up to nt 7650 (plasmid p59-5d7650) or nt 7792 (plasmid p59-5d7792), the transcriptional activity was drastically reduced to 9 and 3% of wildtype, respectively. These results suggest that the downstream sequences from nt 7493 are important for the transcriptional activity of the HPV-59 URR. Internal deletion mutants of the HPV-59 URR were constructed by combining appropriate 5%and 3%-deletion mutants and adding SmaI linkers (Fig. 2B). The transcriptional activities of these deletion mutants are shown in Fig. 4. The transcriptional activity of p59-In7417/7493 was not reduced in comparison to the wildtype, p59-wt-
CAT. Combined with the results from Figs. 3 and 4, the 5%-end of this URR (nt 7149–7493) does not have any recognizable enhancer element for the HPV-59 promoter. However, when we deleted the region between nt 7493 and 7742, CAT activities of p59-In7466/7650, p59-In7544/7650, and p59-In7544/7792 were drastically reduced as shown in Fig. 4. Thus, we carefully compared the URR sequences with known transcription factors response elements. Putative binding sites for transcription factors are present in the region around nt 7296 (AP1), nt 7405 and 7411 (Oct1), nt 7429 and 7450 (YY1), nt 7425 (CREB), and nt 7467 (E2-BSc 1) as shown in Fig. 5, but they do not seem to be essential for the transcription of the
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Fig. 2. Schematic representation of E6 promoter mutants. (A) The filled boxes on the HPV-59 URR denote E2 binding sites (E2-BSs) on known transcriptional transactivators. The deletion end-points of the mutant series are aligned with the sequence. Numbers indicate the nucleotide position of 5% starting points of deletion mutants. (B) Internal deletion mutants contain a SmaI linker in deletion end-points. Deleted regions are shown with dashed lines.
HPV-59 E6 promoter. The region between nt 7493 and 7742 seems to be necessary for the basic transcription of HPV-59 URR. The transcriptional activities of the deletion mutants p59In7466/7650 and p59-In7544/7792 were decreased 14 and 100 fold, respectively, compared to the wildtype p59-wtCAT. In addition, p59-In7544/ 7650 shows about 2-fold reduction in transcriptional activity. When the distal region of nt 7742–7880 was deleted (p59-In7742/7792 and p59-In7742/7880), there was not much difference in transcriptional activity compared to that of the wildtype. Judging from the above data and comparing to the HPV-11 URR (Chin et al., 1989), we divide HPV-59 URR into three parts (Fig. 5), i.e. the distal (nt 7149 – 7493), the central (nt 7493–7742) and the proximal (nt 7742 – 7748) region. In these three regions, the central 249 bp region between nt 7493 and 7742 harbors the important enhancer elements of the HPV-59 URR. In particular, the two sub-regions (nt 7466 and 7544, 7650 and 7742) are important for transcription. A number of recognizable binding sites for cellular transcription factors are present in
these regions (Fig. 5), i.e. five NF1 sites, an AP1 site, two Oct1 sites and one site each for GRE and PVF. The HPV-59 URR harbors four E2-BSs at nt 7467 (E2-BSc 1), nt 7810 (E2-BSc 2), nt 7887 (E2-BSc 3), and nt 7 (E2-BS c 4) (Fig. 2A). To identify the role of each E2-BS in the HPV-59 E6 promoter transcription, 5%-deletion mutants were cotransfected with the HPV-59 E2 expression plasmid (pRc59E2). HPV E2 protein repressed the transcriptional activity of wild type HPV-59 E6 promoter (p59-wtCAT) by 30% (Figs. 1 and 3). The transcriptional activities of p59-5d7307 which had intact E2-BS sequences were repressed to 30% of wildtype in the presence of HPV-59 E2 protein (Fig. 3). When E2-BSc 1 was deleted in plasmid p59-5d7493, the transcriptional activity of this 5%-deletion mutant was reduced to 15% of wildtype (p59-wtCAT) in the presence of the HPV-59 E2 protein. In the case of p59-In7742/7880, in which E2-BSc 2 was deleted, the transcriptional activity was not much different from that of p59In7742/7792, in which no E2-BS was deleted. It was known that the E2 protein acts as a repressor
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Fig. 3. Transcriptional activities of 5% deletion mutants of HPV-59 URR-E6 promoter. One microgram of each 5% deletion mutant DNA with or without 1 mg of HPV-59 E2 expression plasmid was transfected into C33A cells. (B) Relative CAT activity of 5% deletion mutants of the HPV-59 URR-E6 promoter is presented in the absence or presence of HPV-59 E2 expression plasmid, pRc59E2.
by preventing the formation of an initiation complex with TFIID or SP1 at the E6 promoter (Tan et al., 1994; Demeret et al., 1994; Dong et al., 1994). From the above results, we conclude that E2-BSc 3 and c 4 may be more important than E2-BSc 1 and c2 in HPV-59 for its transcriptional regulation because the SP1 binding site and TATA box were closely located to E2-BSc 3 and c4 in the URR.
3.3. Identification of nuclear factors binding to the HPV-59 URR Viral transcriptional regulation is basically mediated by the interaction of trans-acting factors with cis-enhancer elements. Several DNA probes spanning the HPV-59 URR were constructed to examine the trans-acting factors binding to the URR, as shown in Fig. 5. Gel retardation assays were performed using HeLa nuclear extracts and these DNA probes. The central region (nt 7493 – 7742) of URR played an important role in the function of the HPV-59 E6 promoter, as shown in CAT assays (Figs. 3 and 4). We performed binding assays with two DNA fragments (nt 7493 – 7619 and nt 7619–
7742) in this region (Fig. 6). Transcription factors AP1 and Oct1 bound to this central region, whereas the transcription factors NF1 and GRE did not bind to this region. The AP1 consensus sequence is located around nt 7603 (5%TTAGTCA-3%) and there are two consensus sequences of the Oct1 (NFA) binding site at nt 7638 (5%-ACTTGCAT-3%) and nt 7713 (5%-AATTGCAT-3%) in this region. More experiments such as Dnase I footprinting should be carried out to find out the precise binding sites of AP1 and Oct1. Even though there are five consensus sequences of NF1 binding sites in this region, they are half sites (5%-TTGGC-3%). Because the half site of NF1 has a low affinity for NF1, we might not be able to detect retarded complexes on EMSA. The probe 7493-7619 might be complexed with unknown cellular factors (Fig. 6A). This DNA fragment was not competed out by adding known competitor NF1 or AP1 DNA sequences. MSPF and PEF-1 have been known to bind to the HPV-16 enhancer (List et al., 1994; Sibbet et al., 1995). We searched for consensus binding sequences of MSPF and PEF in this central region of HPV-59 URR in vain. Therefore, this central region may contain a binding site for unknown cellular factors.
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Fig. 4. Transcriptional activities of internal deletion mutants of HPV-59 URR-E6 promoter. One microgram of each internal deletion mutant DNA with or without 1 mg of HPV-59 E2 expression plasmid was transfected into C33A cells. Relative CAT activity of internal deletion mutants of the HPV-59 URR-E6 promoter is presented in the absence or presence of HPV-59 E2 expression plasmid, pRc59E2.
The gel-retardation assays resulted in the detection of an SP1 binding in the proximal region (nt 7792–7748) of the HPV-59 URR. As shown in Fig. 7, purified SP1 protein strongly bound to the nt 7880–7748 fragment, and this shifted DNA band was disrupted by adding an SP1 site competitor DNA. These results suggest that the transcription factor SP1 can bind putative SP1 binding sites around nt 7880 in the HPV-59 URR. Although the HPV-59 URR sequence harbors GRE and TFIID binding sites in this distal region, these factors did not seem to bind to this region (data not shown).
4. Discussion To understand the mechanism of tumor induction by HPVs, it is essential to know the mechanisms of the viral early gene regulation. Several promoters within the URR could modulate the regulation of early gene expression, e.g. the HPV E6 promoter. Data have shown that the transcription of the E6 promoter is modulated by cis-elements contained within HPV URRs (Butz and Hoppe-Seyler, 1993). The viral and cellular fac-
tors regulating the transcription of HPV-59, however, have not been characterized. This prompted us to identify the cellular and/or viral factors mediating the regulation of gene expression in the HPV-59 URR as well as the cis-elements responsible for the enhancing effect of the E6 promoter activity. The BPV1 E2 protein has been shown to act as a transactivator on heterologous promoters by binding to its recognition sites (E2-BSs). A synergistic effect between the E2-BSs of BPV1 has been demonstrated (Li et al., 1989). The E2C protein, which is encoded by the 3%-half of the E2 ORF, acts as a repressor on the BPV1 E6 promoter by competing with the full-length E2 protein for the E2-BSs (Bernard et al., 1989; Chin et al., 1989; Romanczuk et al., 1990; Monini et al., 1991). In the genital HPV types, the repression of the E6 promoter by the E2 protein is thought to be due to the E2 protein displacing cellular factors at the two proximal E2-BSs located adjacent to the TATA box (Tan et al., 1992, 1994; Demeret et al., 1994; Dong et al., 1994). Our results demonstrate that the HPV-59 E2 protein acts as a repressor on the transcription of its homologous E6 promoter, whereas it retains a transactivator activity on the
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Fig. 5. Schematic representation of HPV-59 URR-E6 promoter and probes for EMSA. The HPV-59 URR-E6 promoter is divided into three parts: distal, central, and proximal regions. Symbolic representation of the putative cellular factor binding sites is shown. The arrow indicates the direction of transcription of the E6 promoter. Probes scanning a full-region of the URR-E6 promoter are designated three DNA fragments.
BPV1 E6 promoter (Fig. 1). Previous studies have demonstrated that the TATA box and its contiguous E2-BSs are very important for maximal repression of the E6 promoter (Dostatni et al., 1991; Tan et al., 1994). This dual function of the HPV-59 URR is analogous to the URRs of other HPV types. Using deletion mutants, we demonstrated that the constitutive enhancer of the HPV-59 E6 promoter is located within the central 249 bp fragment (nt 7493–7742) of the URR. From analysis of internal deletion mutants (p59-In7466/7650, p59-In7544/7792 in Fig. 4), the region from nt 7466 to 7742 served as an important role for the transcription of E6 promoter. However, we could not detect any recognizable consensus binding sites for cellular transcription factors except AP1 sites. It is suggested that unknown cellular factors may bind to the nt 7493 – 7619 region and may play an important role for the maximal transcription of E6 promoter. AP1 has been identified as a transcriptional factor mediating transcriptional induction by phorbol esters. In previous reports, the HPV E6
promoter is inducible by phorbol esters and contains the specific AP1 binding sites (tetradecanoyl phorbol acetate (TPA)-responsive element (TRE)) (Chan et al., 1990; Butz and Hoppe-Seyler, 1993; Kyo et al., 1995). AP1 binds to the HPV-59 URR in the central region. Interestingly, the central region of HPV-59 URR has an AP1 binding site (5%-TTAGTCA-3%). To further analyze this AP1 element, we tested the inducibility of HPV-59 E6 promoter activity by TPA using CAT assays. The results (data not shown) indicated only a slight activation of the transcription from the HPV-59 E6 promoter by TPA, pointing to the possible involvement of other cellular factors in the function of the AP1 element in the central region. The enhancer activity of the HPV-18 URR was shown to be generated by a functional synergism between a centrally located AP1 element and unidentified cis-elements present in the 5%-flank of the enhancer (Butz and Hoppe-Seyler, 1993). Kyo et al. (1995) also reported that AP1 synergistically activated the HPV-31b enhancer together with either of two novel factors (NF1- like factor and KRF1-like factor). Another explanation is that the
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Fig. 6. EMSA of the central region of HPV-59 URR-E6 promoter. The radiolabeled probe used in each experiment is denoted at the top of the figure. Five micrograms of HeLa nuclear extracts was added with 10 fm of 32P-labeled DNA probe. Also shown is the addition of the various competitors (2 pm), namely the consensus NFI oligonucleotides, AP1 oligonucleotides, Oct1 oligonucleotides, SP1 oligonucleotides, and GRE oligonucleotides. The HindIII linker (10 bp; 5%-CCAAGCTTGG-3%) was used as the non-specific competitor oligonucleotides. The filled-arrows indicate the positions of Oct1 and AP1 shifts. The free DNA probes are shown as open-arrows. (A) EMSA of probe 7493–7619. (B) EMSA of probe 7619 – 7742.
enhancer activity of AP1 in the central region may be strongly dependent on cell-type. The degree of the binding activity of AP1 to its consensus sequence (Kyo et al., 1995), as well as the post-transcriptional modification or protein turnover of AP1 (Offord et al., 1993) have been shown to influence the enhancer activity when different cell types are used. The proximal region of the HPV-59 URR did not significantly influence the transcriptional activity of HPV-59 E6 promoter as demonstrated by deletion mutant analyses (Fig. 3). This contrasted with a strong binding of purified SP1 protein to the proximal fragment of the URR (Fig. 7). This binding of cellular SP1 to HPV-59 differs from the HPV-18 aberrant SP1 element (5%-GGGAGT3%) which is important for the activity of the
HPV-18 URR in cells of epithelial origin (HoppeSeyler and Butz, 1992; Butz and Hoppe-Seyler, 1993). Furthermore, the mutation of the SP1 element led to the slight reduction of HPV-18 E6 promoter activity when tested in the cervical carcinoma cell line C33A (Hoppe-Seyler and Butz, 1992). We cannot exclude the possibility that the enhancer activity of the HPV-59 SP1 element is dependent on cell-type specificity.
Acknowledgements We thank Dr Louise T. Chow for providing C33A cells and plasmids used in this study. This study was supported in part by grants from the Korea Science and Engineering Foundation for
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Fig. 7. EMSA of the proximal region of the HPV-59 URR-E6 promoter. Sequence-specific binding of purified SP1 to the 5% portion of HPV-59 URR-E6 promoter. Five micrograms of HeLa nuclear extracts or 1 mg of purified SP1 was added with 10 fm of 32P-labeled DNA probe in the presence or absence of the specific SP1 competitors (2 pm). The filled-arrow indicates the positions of SP1 shifts. The free DNA probes are shown as open-arrows.
Korean-German Cooperative Science Program, the Genetic Engineering Research Grant from the Ministry of the Republic of Korea, and the Science Research Center for Cell Differentiation at Seoul National University. References Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (Eds.) (1990) Current protocols in molecular biology. Wiley Interscience, New York. Bernard, B.A., Bailly, C., Lenoir, M.C., Darmon, M., Thierry, F. and Yaniv, M. (1989) The human papillomavirus type 18 (HPV18) E2 gene product is a repressor of the HPV18 regulatory region in human keratinocytes. J. Virol. 63, 4317 – 4324. Butz, K. and Hoppe-Seyler, F. (1993) Transcriptional control of human papillomavirus (HPV) oncogene expression: composition of the HPV type 18 upstream regulatory region. J. Virol. 67, 6476–6486.
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Chan, W.-K., Klock, G. and Bernard, H.U. (1989) Progesterone and glucocorticoid response elements occur in the long control regions of several human papillomaviruses involved in anogenital neoplasia. J. Virol. 63, 3261 – 3269. Chan, W.-K., Chong, T., Bernard, H.-U. and Klock, G. (1990) Transcription of the transforming genes of the oncogenic human papillomavirus-16 is stimulated by tumor promoters through AP1 binding sites. Nucleic Acids Res. 18, 763 – 769. Chin, M.T., Broker, T.R. and Chow, L.T. (1989) Identification of a novel constitutive enhancer element and an associated binding protein: implications for human papillomavirus type 11 enhancer regulation. J. Virol. 63, 2967 – 2976. Choe, J., Vaillancourt, P., Stenlund, A. and Botchan, M. (1989) Bovine papillomavirus type 1 encodes for two forms of a transcriptional repressor: structural and functional analysis of new viral cDNAs. J. Virol. 63, 1743 – 1755. de Roda Husman, A.M., Walboomers, J.M., Meijer, C.J., Risse, E.K., Schipper, M.E., Helmerhorst, T.M., Bleker, O.P., Delius, H., van den Brule, A.J. and Snijders, P.J. (1994) Analysis of cytomorphologically abnormal cervical scrapes for the presence of 27 mucosotropic human papillomavirus genotypes, using polymerase chain reaction. Int. J. Cancer 56, 802 – 806. de Villiers, E.-M. (1994) Human pathogenic papillomavirus types: An update. In: H. zur Hausen (Ed.) Current Topics in Microbiology and Immunology, Springer Verlag, Berlin, 186, pp. 1 – 12. Demeret, C., Yaniv, M. and Thierry, F. (1994) The E2 transcriptional repressor can compensate for SP1 activation of the human papillomavirus type 18 early promoter. J. Virol. 68, 7075 – 7082. Dostani, N., Lambert, P., Sousa, R., Ham, J., Howley, P. and Yaniv, M. (1991) The functional BPV-1 E2 trans-activating protein can act as a repressor by preventing formation of the initiation complex. Genes Dev. 5, 1657 – 1671. Dong, G., Broker, T.R. and Chow, L.T. (1994) Human papillomavirus type 11 E2 protein repress the homologous E6 promoter by interfering with the binding of host transcription factors to adjacent elements. J. Virol. 68, 1115 – 1127. Gorman, C.M., Moffat, L.F. and Howard, B.H. (1982) Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2, 1044 – 1051. Hoppe-Seyler, F. and Butz, K. (1992) Activation of human papillomavirus type 18 E6 – E7 oncogene expression by transcription factor Sp1. Nucleic Acids Res. 20, 6701 – 6706. Kyo, S., Tam, A. and Laimins, L.A. (1995) Transcriptional activity of human papillomavirus type 31b enhancer is regulated through synergistic interaction of AP1 with two novel cellular factors. Virology 211, 184 – 197. Li, R., Knight, J., Bream, G., Stenlund, A. and Botchan, M. (1989) Specific recognition nucleotides and their DNA context to determine the affinity of E2 protein for 17 binding sites in the BPV-1 genome. Genes Dev. 3, 510 – 526.
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Sanger, F., Nicklen, S. and Coulston, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463 – 5467. Sibbet, G.J., Cuthill, S. and Campo, M.S. (1995) The enhancer in the long control region of human papillomavirus type 16 is up-regulated by PEF-1 and down-regulated by Oct-1. J. Virol. 69, 4006 – 4011. Tan, S.-H., Gloss, B. and Bernard, H.-U. (1992) During negative regulation of the human papillomavirus-16 E6 promoter, the viral E2 protein can displace Sp1 from a proximal promoter element. Nucleic Acids Res. 20, 251 – 256. Tan, S.-H., Leong, L.E.-C. Walker, P.A. and Bernard, H.-U. (1994) The human papillomavirus type 16 E2 transcription factor binds with low cooperativity to two flanking sites and represses the E6 promoter through displacement of Sp1 and TFIID. J. Virol. 68, 6411 – 6420. von Knebel Doeberitz, M., Rittmuller, C., zur Hausen, H. and Durst, M. (1992) Inhibition of tumorigenicity of cervical cancer cells in nude mice by HPV E6-E7 anti-sense RNA. Int. J. Cancer. 51, 831-834. Yang, Y.-C., Okayama, H. and Howley, P.M. (1985) Bovine papillomavirus contains multiple transforming genes. Mol. Cell. Biol. 2, 1030 – 1034. zur Hausen, H. (1987) Papillomaviruses in human cancer. Cancer 59, 1692 – 1696. zur Hausen, H. (1991a) Human papillomaviruses in the pathogenesis of anogenital cancer. Virology 184, 9 – 13. zur Hausen, H. (1991b) Viruses in human cancers. Science 254, 1167 – 1173.
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Identification of cis-regulatory elements in the upstream regulatory region of human papillomavirus type 59 Jaerang Rhoa, Soyoung Leea, Ethel-Michele de Villiersb, Joonho Choea,* a
Department of Biological Sciences, Korea Ad6anced Institute of Science and Technology, Taejon 305 -701, South Korea b Di6ision Tumour6irus-Characterization, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany Received 14 August 1996; revised 16 October 1996; accepted 16 October 1996
Abstract Human papillomavirus type 59 (HPV-59) was cloned from a vulvar intraepithelial neoplasia and the complete nucleotide sequence was determined. This virus is closely related to HPV-18 and -39 (60% homology in nucleotide sequence) and is grouped with the genital HPV types. In the present paper, we demonstrate that the HPV-59 E2 transactivator represses its E6 promoter-mediated transcription. We have also analyzed cis-regulatory elements in the upstream regulatory region (URR) of HPV-59 using chloramphenicol acetyl transferase assays as well as electrophoresis mobility shift assays (EMSA). The results allow for a subdivision of the HPV-59 URR into three regions of activity: distal (nt 7149–7493), central (nt 7493–7742), and proximal (nt 7742 – 7748). In particular, the 250 bp (nt 7493–7742) of the central region plays an important role as a constitutive enhancer element for the maximal transcription of the E6 promoter. Our results suggest that the transcription factors AP1, Oct1, SP1 and unidentified factors bind to the HPV-59 E6 promoter region, whereas NF1, GRE and TFIID fail to bind despite the presence of putative binding sites in the DNA sequence. © 1997 Elsevier Science B.V. Keywords: CAT assay; EMSA; E6 promoter; HPV-59; Upstream regulatory region
1. Introduction Human papillomaviruses (HPVs) are causative agents of a variety of benign and malignant lesions in humans (de Villiers, 1994; zur Hausen, 1991a). Experimental and epidemiological data * Corresponding author. Tel.: + 82 42 8694020; fax: + 82 42 8695260; e-mail: [email protected]
indicate a higher oncogenic potential of certain HPV types associated with lesions such as cervical intraepithelial neoplasia (CIN) or carcinomas. So far, more than 100 HPV types have been identified, including 77 of which the genomes have been characterized (de Villiers, 1994). A large number of these HPV types are closely associated with anogenital carcinomas. In particular, HPV16 and -18 are most frequently present in lesions
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progressing towards malignancy (zur Hausen, 1987) and are grouped as ‘high-risk HPVs’. Other HPV types are similarly associated with malignant progression as such, but are not so frequently detected in populations as in the case of HPV-16 and -18. It is important to characterize these other highly oncogenic HPVs in order to understand whether their low frequency is attributable to the mechanism of their oncogenicity and/or their tissue specificity. Previously we have cloned HPV-59 from a vulvar intraepithelial neoplasia and determined its complete nucleotide sequence (Rho et al., 1994). By comparing the HPV-59 DNA sequence to those of all other HPVs, HPV-59 shares the highest nucleotide homology with HPV-18 (71%), HPV-45 (70%) and HPV-39 (69%), all three belonging to the high-risk group (de Villiers, 1994). HPV-59 DNA was detected in CIN I or CIN II lesions in a large scale studies of analyzed clinical samples (Matsukura and Sugase, 1995; de Roda Husman et al., 1994). HPV-59 is present in populations throughout the world, but is nevertheless only detectable in a small percentage of genital lesions. It has, in addition, been associated with lesions on muco-epithelial junctions (Roy-Burman and de Villiers, unpublished data) as well as in oral lesions. Due to its apparent different biological behaviour from already characterized HPVs, it was of interest to us to analyze the regulation of gene expression in HPV-59. The persistent expression of specific HPV viral transcripts and their proteins has been shown to be necessary for the maintenance of malignant cell growth (von Knebel Doeberitz et al., 1992). In particular, gene products of the E6, E7 open reading frames (ORF) in HPV and E5 ORF in bovine papillomavirus type 1 (BPV1) have been identified as having transforming functions (zur Hausen, 1991b). Transcripts coding for E6, E7 and E5 are initiated from the upstream regulatory region (URR) of the HPV genome, just upstream of the E6 ORF (Romanczuk et al., 1990). The initiation of transcription from the URR is controlled by cellular factors and viral proteins which interact with cellular and viral enhancer elements (Chin et al., 1989; Butz and Hoppe-Seyler, 1993). In addition, HPV URR contains sequences neces-
sary for the regulation of viral DNA replication (Russell and Botchan, 1995) and late gene expression (Choe et al., 1989). The URR therefore plays a key role in regulating the gene functions necessary for the malignant transformation of cells. In this study, we tried to identify transcriptional control elements located in the HPV-59 URR. By sequence alignment, the HPV-59 URR shares the highest homology with the URRs of HPV-18 (62%) and HPV-39 (63%) (Rho et al., 1994). Computer analyses revealed similar characteristics for the HPV-59 URR as previously described for the HPV-16, -18 and -33 (Chan et al., 1989), e.g. a number of consensus sequences for binding sites of transcription factors such as GRE (GGTACANNNTGTTCT), SP1 (GGCGGG) or NF1 (TTGGCA), as well as four E2 binding sites (E2-BS, ACCN6GGT). The positions of the E2BS are highly conserved as in other genital HPVs which have two E2-BSs located close to the TATA box of the E6 promoter. The E2 protein of the HPVs can function either as a transactivator or as a repressor (Choe et al., 1989). It acts as a repressor by preventing the formation of an initiation complex with TFIID or SP1 at the E6 promoter (Tan et al., 1992, 1994; Demeret et al., 1994; Dong et al., 1994). It was of interest to us to test whether these binding sites are utilized by cellular transcription factors. We report here the detailed analysis of the HPV-59 URR using the chloramphenicol acetyltransferase (CAT) assay and gel retardation assay with nuclear extracts. Despite the structural similarity to other HPV URRs, the regulation of early gene expression is modulated differently in HPV59 URR.
2. Materials and methods
2.1. Plasmids The HPV-59 URR DNA was amplified by the polymerase chain reaction (PCR) using the forward primer 59c 7149 (5%-GTTAAGCTTTTGTATGTGTGCATG-3%) at nt 7149 and backward primer 59c 48 (5%-GCGTGCCTTGCTGCAGCCCTTTTCTTTTC-3%) at nt 48 and subcloned
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into the pUC19 vector. This amplified product containing the HPV-59 URR and the E6 promoter was subcloned into the upstream region of the CAT gene of pCAT-Basic reporter vector (Promega) and named p59-wtCAT. To construct a HPV-59 E2 expression plasmid, the HPV-59 E2 ORF (nt 2719 – 3894) was amplified using a forward primer 59 c2719 (5%-GATAAGCTTGGAACGAGGAAGAG-3%) at nt 2719 and a backward primer 59 c3804 (5%-CCTCATCTAGACAATATAGCAGCAG-3%) and subcloned into the pRc/CMV expression vector (Invitrogen). It was named pRc59E2. For transient transfection assays pSV2CAT was used as a positive control (Gorman et al., 1982). The p2CAT DNA contains the BPV1 URR and P2 promoter region attached to a CAT gene (Rho et al., 1993). NDE2, which contains the E2 ORF of BPV1, is a derivative of pC59 E2 (Yang et al., 1985).
2.2. Generation of deletion mutants To construct 5% deletion mutants of HPV-59 URR, p59-wtCAT was used as a template for PCR. The sense strand primers were synthesized as follows: 5%-GAGTAAGCTTCTGTCCCTTTATTG-3% (59 c7307) at nt 7307, 5%-GTTTAAGCTTACCTTTTTGAAC-3% (59c 7493) at nt 7493, 5%-CATAAGCTTGGTGGCGCCCTAATA-3% (59c7650) at nt 7650, 5%-ACTAAGCTTTTTGTCATTGTTAAG-3% (59c7792) at nt 7792, 5%-AATAAGCTTGGGTGTAACCGAAAAC-3% (59c7880) at nt 7880. Amplification was performed with an antisense primer 59c 48 at 94°C, 53°C, and 72°C for 2 min each and for 30 cycles. Each of the five PCR products was cloned into a pCAT-Basic vector and labeled as p59-5d7307, p59-5d7493, p595d7650, p59-In7792, and p59-In7880, respectively. Deletion mutants from the 3% end of HPV-59 URR were constructed as follows. The plasmid p59-wtCAT was linearized by PstI digestion. The linearized DNA was digested with exonuclease BAL31 at 30°C between 1 and 40 min. After digestion, the DNA was incubated with DNA
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polymerase Klenow fragment and deoxynucleoside triphosphates to generate blunt ends before a PstI-linker was added. The deleted DNA was digested with HindIII/PstI and ligated into pUC19. The deletion endpoint of each mutant was determined by dideoxy-sequencing (Sanger et al., 1977). Internal deletion mutants were generated by combining appropriate pairs of 5%- and 3%-deletion mutants. Deletion mutants from the 5%-end were digested with HindIII and blunt-ends were generated by the DNA polymerase Klenow fragment before a SmaI-linker was added. Deletion mutants from the 3%-end were digested with PstI and blunt-ends were generated with T4 DNA polymerase and SmaI-linkers added. Deletion mutants from the 5%-end were digested with SmaI and PstI and the SmaI/PstI fragments were subcloned into the large fragments of the HindIII/SmaI of the 3%-deletion mutants. Six internal deletion mutants were confirmed by PCR amplification.
2.3. Cell culture and transient transfection assay C33A cervical carcinoma cells (obtained from Dr Louise T. Chow) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 4.5 g glucose/l and 100 U/ml penicillin and 100 mg/ml streptomycin in an atmosphere of 5% CO2 at 37°C. One milligram of the HPV-59 URR-E6 promoter plasmids and 2.5 mg of the E2 expression plasmid (pRc59E2) were transfected, unless otherwise stated. The total amount of DNA (10 mg) was kept constant by adding salmon sperm DNA as a carrier. Approximately 5× 105 cells were transfected by calcium-phosphate coprecipitation as previously described (Rho et al., 1993) with the following modifications. The DNA-calcium precipitate was applied to the cells for 6 h followed by a shock with 15% glycerol for 1 min. The cells were then washed three times with phosphate buffered saline (PBS) and cultured in DMEM/FCS. Thirty six hours after the glycerol shock the cell extracts were prepared as follows: cells were frozen (−70°C) and thawed at 37°C three times. CAT assays were performed for 5 h according to the standard protocol (Ausubel et
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al., 1990), with the amount of cell extract adjusted to keep the chloramphenicol conversion rate within linear range (100 – 200 mg). The percent conversion of chloramphenicol to the acetylated form was quantitated by Phosphor-Imager (Molecular Dynamics). Each experiment was repeated at least three times.
2.4. Gel retardation assay and preparation of DNA probes The electrophoresis mobility shift assay (EMSA) was performed essentially according to the manufacturer’s protocol (Promega). The binding reaction was performed using 5 mg of HeLa nuclear extracts in binding buffer (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCl, pH 7.5, and 50 mg/ml poly (dI-dC)). The samples were preincubated with or without a specific competitor DNA (2 pm) at room temperature for 10 min. The consensus oligonucleotides of binding sites for several transcription factors present in the EMSA assay were purchased from Promega to use as competitor DNA. The sequences of these oligonucleotides are: SP1: 5%-ATTCGATCGGGGCGGGGCGAGC-3%, AP1: 5%-CGCTTGATGAGTCAGCCGGAA-3%, GRE: 5%-TCGACTGTACAGGATGTTCTAGC TACT-3%, CTF/NF1: 5%-CCTTTGGCATGCTGCCAATAT-3%,
with HindIII/ApyI and the 126 bp fragment was isolated. The probe 7619–7742 was generated by digestion of p59-In7742/7792 with ApyI/SmaI and the 123 bp fragment was eluted. The probe 7792-48 was constructed by digestion of p595d7792 with HindIII/PstI and the 152 bp fragment was isolated. All 5% ends of these DNA fragments were labelled with 32P-ATP by T4 DNA kinase.
3. Results
3.1. HPV-59 E2 protein represses the transcription of E6 promoter To identify the function of the HPV-59 E2 protein in the transcription of the HPV-59 E6 promoter, we transfected the p59-wtCAT DNA into C33A cells with different amounts of the HPV-59 E2 expressing plasmid (pRc59E2). HPV-59 E2 expression resulted in a repression of the reporter activity to a level of about 30% of the control activity (Fig. 1). When p2CAT DNA, which contains the BPV1 E6 promoter, was cotransfected with pRc59E2, the transcription activity was rather slightly (1.4-fold) activated. BPV1 E2 expression also activated its own E6 promoter-mediated transcription by about 3.4-fold. Therefore, the HPV-59 E2 protein behaves differently from the BPV1 E2 protein in regulating its own E6 promoter.
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P-labeled DNA probe (10 fm) was added to each reaction. The reaction mixture was incubated at room temperature for 20 min, after which gel loading buffer (25 mM Tris – HCl, pH 7.5, 0.02% bromophenol blue, 0.02% xylene cyanol, 4% glycerol) was added. The reaction was analyzed by electrophoresis in a 4% polyacrylamide gel (80:1, acrylamide:bisacrylamide; 2.5% glycerol; 0.5×TBE (45 mM Tris – HCl, pH 8.0, 45 mM boric acid, 1 mM EDTA)). To make DNA probes of HPV-59 URR for EMSA, we constructed several DNA fragments spanning the URR as follows: the probe 7493– 7619 was constructed by digesting p59-5d7493
3.2. Analysis of enhancer elements in the HPV-59 URR To identify enhancer elements in the HPV-59 URR, five deletion mutants from the 5%-end were constructed by PCR amplification as described in Section 2 (Fig. 2A). These plasmids were transfected into C33A cells in the presence or absence of pRc59E2 containing the HPV-59 E2 ORF. As shown on Fig. 3, when the URR was deleted between the 5%-end (nt 7149) and nt 7493 (plasmids p59-5d7307 and p59-5d7493), the transcriptional activity of the URR was not influenced in
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Fig. 1. The functional effect of HPV-59 E2 protein on the HPV-59 E6 and BPV1 E6 promoters. Relative CAT activities of HPV-59 E6 and BPV1 E6 promoters (1 mg of DNA) are shown. The cells were cotransfected with increasing amounts of the HPV-59 E2 or BPV1 E2 expression plasmid, pSV2CAT was transfected as the positive control. Relative CAT activity was defined as the ratio of the percent acetylated chloramphenicol to that obtained with extracts made from cells transfected with p59-wtCAT.
comparison to the full-length URR (p59-wtCAT). However, when the URR was deleted up to nt 7650 (plasmid p59-5d7650) or nt 7792 (plasmid p59-5d7792), the transcriptional activity was drastically reduced to 9 and 3% of wildtype, respectively. These results suggest that the downstream sequences from nt 7493 are important for the transcriptional activity of the HPV-59 URR. Internal deletion mutants of the HPV-59 URR were constructed by combining appropriate 5%and 3%-deletion mutants and adding SmaI linkers (Fig. 2B). The transcriptional activities of these deletion mutants are shown in Fig. 4. The transcriptional activity of p59-In7417/7493 was not reduced in comparison to the wildtype, p59-wt-
CAT. Combined with the results from Figs. 3 and 4, the 5%-end of this URR (nt 7149–7493) does not have any recognizable enhancer element for the HPV-59 promoter. However, when we deleted the region between nt 7493 and 7742, CAT activities of p59-In7466/7650, p59-In7544/7650, and p59-In7544/7792 were drastically reduced as shown in Fig. 4. Thus, we carefully compared the URR sequences with known transcription factors response elements. Putative binding sites for transcription factors are present in the region around nt 7296 (AP1), nt 7405 and 7411 (Oct1), nt 7429 and 7450 (YY1), nt 7425 (CREB), and nt 7467 (E2-BSc 1) as shown in Fig. 5, but they do not seem to be essential for the transcription of the
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Fig. 2. Schematic representation of E6 promoter mutants. (A) The filled boxes on the HPV-59 URR denote E2 binding sites (E2-BSs) on known transcriptional transactivators. The deletion end-points of the mutant series are aligned with the sequence. Numbers indicate the nucleotide position of 5% starting points of deletion mutants. (B) Internal deletion mutants contain a SmaI linker in deletion end-points. Deleted regions are shown with dashed lines.
HPV-59 E6 promoter. The region between nt 7493 and 7742 seems to be necessary for the basic transcription of HPV-59 URR. The transcriptional activities of the deletion mutants p59In7466/7650 and p59-In7544/7792 were decreased 14 and 100 fold, respectively, compared to the wildtype p59-wtCAT. In addition, p59-In7544/ 7650 shows about 2-fold reduction in transcriptional activity. When the distal region of nt 7742–7880 was deleted (p59-In7742/7792 and p59-In7742/7880), there was not much difference in transcriptional activity compared to that of the wildtype. Judging from the above data and comparing to the HPV-11 URR (Chin et al., 1989), we divide HPV-59 URR into three parts (Fig. 5), i.e. the distal (nt 7149 – 7493), the central (nt 7493–7742) and the proximal (nt 7742 – 7748) region. In these three regions, the central 249 bp region between nt 7493 and 7742 harbors the important enhancer elements of the HPV-59 URR. In particular, the two sub-regions (nt 7466 and 7544, 7650 and 7742) are important for transcription. A number of recognizable binding sites for cellular transcription factors are present in
these regions (Fig. 5), i.e. five NF1 sites, an AP1 site, two Oct1 sites and one site each for GRE and PVF. The HPV-59 URR harbors four E2-BSs at nt 7467 (E2-BSc 1), nt 7810 (E2-BSc 2), nt 7887 (E2-BSc 3), and nt 7 (E2-BS c 4) (Fig. 2A). To identify the role of each E2-BS in the HPV-59 E6 promoter transcription, 5%-deletion mutants were cotransfected with the HPV-59 E2 expression plasmid (pRc59E2). HPV E2 protein repressed the transcriptional activity of wild type HPV-59 E6 promoter (p59-wtCAT) by 30% (Figs. 1 and 3). The transcriptional activities of p59-5d7307 which had intact E2-BS sequences were repressed to 30% of wildtype in the presence of HPV-59 E2 protein (Fig. 3). When E2-BSc 1 was deleted in plasmid p59-5d7493, the transcriptional activity of this 5%-deletion mutant was reduced to 15% of wildtype (p59-wtCAT) in the presence of the HPV-59 E2 protein. In the case of p59-In7742/7880, in which E2-BSc 2 was deleted, the transcriptional activity was not much different from that of p59In7742/7792, in which no E2-BS was deleted. It was known that the E2 protein acts as a repressor
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Fig. 3. Transcriptional activities of 5% deletion mutants of HPV-59 URR-E6 promoter. One microgram of each 5% deletion mutant DNA with or without 1 mg of HPV-59 E2 expression plasmid was transfected into C33A cells. (B) Relative CAT activity of 5% deletion mutants of the HPV-59 URR-E6 promoter is presented in the absence or presence of HPV-59 E2 expression plasmid, pRc59E2.
by preventing the formation of an initiation complex with TFIID or SP1 at the E6 promoter (Tan et al., 1994; Demeret et al., 1994; Dong et al., 1994). From the above results, we conclude that E2-BSc 3 and c 4 may be more important than E2-BSc 1 and c2 in HPV-59 for its transcriptional regulation because the SP1 binding site and TATA box were closely located to E2-BSc 3 and c4 in the URR.
3.3. Identification of nuclear factors binding to the HPV-59 URR Viral transcriptional regulation is basically mediated by the interaction of trans-acting factors with cis-enhancer elements. Several DNA probes spanning the HPV-59 URR were constructed to examine the trans-acting factors binding to the URR, as shown in Fig. 5. Gel retardation assays were performed using HeLa nuclear extracts and these DNA probes. The central region (nt 7493 – 7742) of URR played an important role in the function of the HPV-59 E6 promoter, as shown in CAT assays (Figs. 3 and 4). We performed binding assays with two DNA fragments (nt 7493 – 7619 and nt 7619–
7742) in this region (Fig. 6). Transcription factors AP1 and Oct1 bound to this central region, whereas the transcription factors NF1 and GRE did not bind to this region. The AP1 consensus sequence is located around nt 7603 (5%TTAGTCA-3%) and there are two consensus sequences of the Oct1 (NFA) binding site at nt 7638 (5%-ACTTGCAT-3%) and nt 7713 (5%-AATTGCAT-3%) in this region. More experiments such as Dnase I footprinting should be carried out to find out the precise binding sites of AP1 and Oct1. Even though there are five consensus sequences of NF1 binding sites in this region, they are half sites (5%-TTGGC-3%). Because the half site of NF1 has a low affinity for NF1, we might not be able to detect retarded complexes on EMSA. The probe 7493-7619 might be complexed with unknown cellular factors (Fig. 6A). This DNA fragment was not competed out by adding known competitor NF1 or AP1 DNA sequences. MSPF and PEF-1 have been known to bind to the HPV-16 enhancer (List et al., 1994; Sibbet et al., 1995). We searched for consensus binding sequences of MSPF and PEF in this central region of HPV-59 URR in vain. Therefore, this central region may contain a binding site for unknown cellular factors.
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Fig. 4. Transcriptional activities of internal deletion mutants of HPV-59 URR-E6 promoter. One microgram of each internal deletion mutant DNA with or without 1 mg of HPV-59 E2 expression plasmid was transfected into C33A cells. Relative CAT activity of internal deletion mutants of the HPV-59 URR-E6 promoter is presented in the absence or presence of HPV-59 E2 expression plasmid, pRc59E2.
The gel-retardation assays resulted in the detection of an SP1 binding in the proximal region (nt 7792–7748) of the HPV-59 URR. As shown in Fig. 7, purified SP1 protein strongly bound to the nt 7880–7748 fragment, and this shifted DNA band was disrupted by adding an SP1 site competitor DNA. These results suggest that the transcription factor SP1 can bind putative SP1 binding sites around nt 7880 in the HPV-59 URR. Although the HPV-59 URR sequence harbors GRE and TFIID binding sites in this distal region, these factors did not seem to bind to this region (data not shown).
4. Discussion To understand the mechanism of tumor induction by HPVs, it is essential to know the mechanisms of the viral early gene regulation. Several promoters within the URR could modulate the regulation of early gene expression, e.g. the HPV E6 promoter. Data have shown that the transcription of the E6 promoter is modulated by cis-elements contained within HPV URRs (Butz and Hoppe-Seyler, 1993). The viral and cellular fac-
tors regulating the transcription of HPV-59, however, have not been characterized. This prompted us to identify the cellular and/or viral factors mediating the regulation of gene expression in the HPV-59 URR as well as the cis-elements responsible for the enhancing effect of the E6 promoter activity. The BPV1 E2 protein has been shown to act as a transactivator on heterologous promoters by binding to its recognition sites (E2-BSs). A synergistic effect between the E2-BSs of BPV1 has been demonstrated (Li et al., 1989). The E2C protein, which is encoded by the 3%-half of the E2 ORF, acts as a repressor on the BPV1 E6 promoter by competing with the full-length E2 protein for the E2-BSs (Bernard et al., 1989; Chin et al., 1989; Romanczuk et al., 1990; Monini et al., 1991). In the genital HPV types, the repression of the E6 promoter by the E2 protein is thought to be due to the E2 protein displacing cellular factors at the two proximal E2-BSs located adjacent to the TATA box (Tan et al., 1992, 1994; Demeret et al., 1994; Dong et al., 1994). Our results demonstrate that the HPV-59 E2 protein acts as a repressor on the transcription of its homologous E6 promoter, whereas it retains a transactivator activity on the
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Fig. 5. Schematic representation of HPV-59 URR-E6 promoter and probes for EMSA. The HPV-59 URR-E6 promoter is divided into three parts: distal, central, and proximal regions. Symbolic representation of the putative cellular factor binding sites is shown. The arrow indicates the direction of transcription of the E6 promoter. Probes scanning a full-region of the URR-E6 promoter are designated three DNA fragments.
BPV1 E6 promoter (Fig. 1). Previous studies have demonstrated that the TATA box and its contiguous E2-BSs are very important for maximal repression of the E6 promoter (Dostatni et al., 1991; Tan et al., 1994). This dual function of the HPV-59 URR is analogous to the URRs of other HPV types. Using deletion mutants, we demonstrated that the constitutive enhancer of the HPV-59 E6 promoter is located within the central 249 bp fragment (nt 7493–7742) of the URR. From analysis of internal deletion mutants (p59-In7466/7650, p59-In7544/7792 in Fig. 4), the region from nt 7466 to 7742 served as an important role for the transcription of E6 promoter. However, we could not detect any recognizable consensus binding sites for cellular transcription factors except AP1 sites. It is suggested that unknown cellular factors may bind to the nt 7493 – 7619 region and may play an important role for the maximal transcription of E6 promoter. AP1 has been identified as a transcriptional factor mediating transcriptional induction by phorbol esters. In previous reports, the HPV E6
promoter is inducible by phorbol esters and contains the specific AP1 binding sites (tetradecanoyl phorbol acetate (TPA)-responsive element (TRE)) (Chan et al., 1990; Butz and Hoppe-Seyler, 1993; Kyo et al., 1995). AP1 binds to the HPV-59 URR in the central region. Interestingly, the central region of HPV-59 URR has an AP1 binding site (5%-TTAGTCA-3%). To further analyze this AP1 element, we tested the inducibility of HPV-59 E6 promoter activity by TPA using CAT assays. The results (data not shown) indicated only a slight activation of the transcription from the HPV-59 E6 promoter by TPA, pointing to the possible involvement of other cellular factors in the function of the AP1 element in the central region. The enhancer activity of the HPV-18 URR was shown to be generated by a functional synergism between a centrally located AP1 element and unidentified cis-elements present in the 5%-flank of the enhancer (Butz and Hoppe-Seyler, 1993). Kyo et al. (1995) also reported that AP1 synergistically activated the HPV-31b enhancer together with either of two novel factors (NF1- like factor and KRF1-like factor). Another explanation is that the
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Fig. 6. EMSA of the central region of HPV-59 URR-E6 promoter. The radiolabeled probe used in each experiment is denoted at the top of the figure. Five micrograms of HeLa nuclear extracts was added with 10 fm of 32P-labeled DNA probe. Also shown is the addition of the various competitors (2 pm), namely the consensus NFI oligonucleotides, AP1 oligonucleotides, Oct1 oligonucleotides, SP1 oligonucleotides, and GRE oligonucleotides. The HindIII linker (10 bp; 5%-CCAAGCTTGG-3%) was used as the non-specific competitor oligonucleotides. The filled-arrows indicate the positions of Oct1 and AP1 shifts. The free DNA probes are shown as open-arrows. (A) EMSA of probe 7493–7619. (B) EMSA of probe 7619 – 7742.
enhancer activity of AP1 in the central region may be strongly dependent on cell-type. The degree of the binding activity of AP1 to its consensus sequence (Kyo et al., 1995), as well as the post-transcriptional modification or protein turnover of AP1 (Offord et al., 1993) have been shown to influence the enhancer activity when different cell types are used. The proximal region of the HPV-59 URR did not significantly influence the transcriptional activity of HPV-59 E6 promoter as demonstrated by deletion mutant analyses (Fig. 3). This contrasted with a strong binding of purified SP1 protein to the proximal fragment of the URR (Fig. 7). This binding of cellular SP1 to HPV-59 differs from the HPV-18 aberrant SP1 element (5%-GGGAGT3%) which is important for the activity of the
HPV-18 URR in cells of epithelial origin (HoppeSeyler and Butz, 1992; Butz and Hoppe-Seyler, 1993). Furthermore, the mutation of the SP1 element led to the slight reduction of HPV-18 E6 promoter activity when tested in the cervical carcinoma cell line C33A (Hoppe-Seyler and Butz, 1992). We cannot exclude the possibility that the enhancer activity of the HPV-59 SP1 element is dependent on cell-type specificity.
Acknowledgements We thank Dr Louise T. Chow for providing C33A cells and plasmids used in this study. This study was supported in part by grants from the Korea Science and Engineering Foundation for
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Fig. 7. EMSA of the proximal region of the HPV-59 URR-E6 promoter. Sequence-specific binding of purified SP1 to the 5% portion of HPV-59 URR-E6 promoter. Five micrograms of HeLa nuclear extracts or 1 mg of purified SP1 was added with 10 fm of 32P-labeled DNA probe in the presence or absence of the specific SP1 competitors (2 pm). The filled-arrow indicates the positions of SP1 shifts. The free DNA probes are shown as open-arrows.
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