CCAAT Displacement Protein Binds to and ... - Journal of Virology

JOURNAL OF VIROLOGY, May 1999, p. 4220–4229 0022-538X/99/$04.0010 Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 73, No. 5

CCAAT Displacement Protein Binds to and Negatively Regulates Human Papillomavirus Type 6 E6, E7, and E1 Promoters WANDONG AI, ESRA TOUSSAINT,

AND

ANN ROMAN*

Department of Microbiology and Immunology, Indiana University School of Medicine, and Walther Cancer Institute, Indianapolis, Indiana 46202-5120 Received 9 September 1998/Accepted 16 February 1999

Expression of human papillomavirus genes increases as the target cell, the keratinocyte, differentiates. CCAAT displacement protein (CDP) is a cellular protein which has been shown in other cell types to negatively regulate gene expression in undifferentiated cells but not in differentiated cells. We have previously shown that a 66-bp purine-thymidine-rich sequence (the 66-mer) binds CDP and negatively regulates the human papillomavirus type 6 (HPV-6) E6 promoter (S. Pattison, D. G. Skalnik, and A. Roman, J. Virol. 71:2013–2022, 1997). Cotransfection experiments with a plasmid expressing luciferase from the HPV-6 E6, E7, or E1 regulatory region and a plasmid carrying the CDP gene indicate that CDP represses transcription from all three HPV-6 promoters. Using electrophoretic mobility shift assays (EMSAs), we have shown that CDP binds HPV-6 both upstream and downstream of the E6, E7, and E1 transcription initiation start sites. Furthermore, when keratinocytes were induced to differentiate, all three promoter activities increased. Consistent with this, immunoblotting and EMSAs revealed that endogenous nucleus CDP and, correspondingly, DNA binding activity decreased when keratinocytes were induced to differentiate. The elevated promoter activities were abrogated by exogenously transfected CDP. Our data demonstrate that CDP fulfills the requirement of a differentiation-dependent negative regulator that could tie the HPV life cycle to keratinocyte differentiation. epithelium and allow visualization of gene expression and DNA amplification in different layers through immunohistochemistry and nucleic acid hybridization. However, the protocol is quite time-consuming, requiring approximately 2 weeks for total stratification (37) and 12 days for peak expression of late HPV RNA (41). In contrast, suspension in methylcellulose allows rapid induction of differentiation with loss of colonyforming ability (27), gain of expression of involucrin in 100% of the cells, and peak induction of HPV late gene expression within 24 h (46). The shift to high levels of calcium yields large numbers of stratified cells undergoing differentiation in the presence of proliferating cells (18, 20, 28, 47). Within 48 h of incubation in high levels of calcium or methylcellulose, a switch in the mode of viral replication can be detected (20). Although HPVs contain a double-stranded genome, only one of the DNA strands is transcribed. For the low-risk viruses, represented by two closely related HPVs, HPV-6 and HPV-11, three early promoters have been identified: the E6 promoter, which initiates transcription within the LCR at nucleotide (nt) 90 (P90); the E7 promoter, initiating transcription in the middle of the E6 open reading frame (ORF) at nt 270 (P270) (44, 49); and the E1 promoter (P680), initiating transcription at nt 680 within the E7 ORF (10, 29). Using in situ hybridization of sections from condyloma acuminata, Stoler et al. (50) showed that the HPV-6 and -11 E6 and E7 transcripts, as well as the E1∧E4 spliced transcript, were barely detectable in basal cells. These transcripts increased in the differentiating epithelium, with the E1∧E4 transcript being the most predominant, indicating that the E1 promoter is a differentiation-specific promoter. Although agreeing with E1 promoter up-regulation upon differentiation, Iftner et al. (26) showed a different expression pattern for the E6 and E7 transcripts. From their analysis of HPV-6-positive anogenital condylomata, E6 transcripts were restricted to the undifferentiated cell layer of the epithelium, and E7 was restricted to the suprabasal cell layer.

Human papillomaviruses (HPVs) are a large group of human pathogens that cause epithelial hyperproliferative lesions. They are small DNA viruses containing a circular, doublestranded DNA genome, approximately 8 kb in length. Over 100 different HPV genotypes have been identified (62), and approximately one-third of them are considered genital and mucosal types because they mainly infect genital and mucosal sites. The HPVs which infect the genital tract are further classified into high-risk and low-risk types. High-risk HPVs are found predominantly in malignancies, such as cervical cancer, while low-risk HPVs are generally found in benign lesions, such as condyloma acuminata (genital warts). All HPVs have essentially the same genome organization, which includes the early region, encoding the nonstructural viral proteins E1 through E7; the late region, encoding the two structural proteins L1 and L2; and the long control region (LCR), located between the termination codon of L1 and the initiation codon of E6, containing cis elements which regulate viral DNA replication and transcription. The HPV life cycle is closely linked to the differentiation program of its host cell, the keratinocyte. HPV DNA amplification and late gene transcription occur mainly in differentiated keratinocytes, although a low level of late gene transcripts has been documented in undifferentiated keratinocytes (8, 41). Three different systems have been used in vitro to examine these differentiation-dependent viral activities: organotypic or raft cultures (9, 16, 21, 22, 25, 38, 39, 41), suspension in methylcellulose (20, 46), or incubation of submerged cultures in media containing high levels of calcium (4, 20, 28). The organotypic cultures most faithfully represent the differentiating * Corresponding author. Mailing address: Department of Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202-5120. Phone: (317) 274-7275. Fax: (317) 274-4090. E-mail: [email protected]. 4220

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TABLE 1. Primers used in PCR to amplify regulatory regions

E7R-1 E7p E7R-2 E7R-3 E1R-1

Nucleotide position

Primer sequencea

Fragment 59

39

59

39

59-GCCTGCAGATGGAAAGGTGCAAATGC 59-GCCTGCAGATGGAAAGGTGCAAATGC 59-TCTAGAGGCGGCTATCCATATGCAG 59-GGCTGCAGACATCTTAGACGTGC 59-GCCTGCAGATGCATGGAAGACATG

59-GCAAGCTTGTTGTCCAGCAGTGTAG 59-AAGCTTCGAAACAGGACCTTTAGG 59-AAGCTTGTTTAGTTTCTTCTTCAACTG 59-GCAAGCTTGTTGTCCAGCAGTGTAG 59-GCAAGCTTCGTTGTTAGGTCTTCG

100 100 268 383 528

527 267 382 527 829

Length in HPV genome (bp)

428 168 115 145 302

a Underlining shows the restriction site used for cloning. At the 59 end of 59 primers, a PstI site (59-CTGCAG) was introduced, except for E7R-2, for which an XbaI site (59-TCTAGA) was used. At the 59 end of 39 primers, a HindIII site (59-AAGCTT) was introduced.

Using a sensitive RNase protection assay, DiLorenzo and Steinberg (15) showed that the HPV-11 E6 transcript predominated over the E7 transcript, and E1∧E4 transcript was often undetectable in cultured cells derived from laryngeal papillomas. When these cells were induced to differentiate in organotypic cultures, the E6 and E1∧E4 transcripts selectively increased (15). Using a retroviral vector with the HPV-11 LCR regulating the lacZ gene, Zhao et al. (61) have shown that the AP1, Oct 1, and Sp1 cis elements within the LCR are each required for up-regulation of the E6 promoter during differentiation of keratinocytes on rafts. Recent data indicate that the low-risk E6 and E7 promoters can be independently regulated by viral and cellular transactivators. Using mutational analysis, Rapp et al. (44) showed that the viral E2 protein represses the HPV-6 E6 promoter through the E6 promoter-proximal E2 binding site, while activating the E7 promoter through the most-promoter-distal E2 binding site. Using laryngeal mucosal keratinocytes, DiLorenzo et al. (14) have shown that expression from the HPV-11 E6 promoter is more sensitive than expression from the E7 promoter to repression by E2 and to mutation of the Sp1 binding site adjacent to the E6 promoter-proximal E2 binding site. Moreover, the E7 promoter activity decreased to a greater extent than the E6 promoter activity following mutation of the E6 TATA box. In previous experiments, we showed that a purine-thymidine-rich 66-bp sequence (the 66-mer) located in the 59 end of the HPV-6W50 LCR binds CCAAT displacement protein (CDP) and negatively regulates the HPV-6 E6 early promoter activity, located at the 39 end of the LCR (42). CDP is a 180-kDa protein which is related to the Drosophila melanogaster Cut homeodomain protein (24, 40). Mammalian homologues of Drosophila Cut from human, dog, mouse, and rat cells have been cloned and termed huCut (or CDP), Clox, Cux, and CDP-2, respectively (43, 55, 59). All homologues contain five conserved regions: a coiled-coil region, three Cut repeats, and one homeodomain (24). The three Cut repeats and the homeodomain have broad DNA binding activity (2, 5, 23). CDP functions as a transcriptional repressor in undifferentiated cells and is not functional in differentiated cells (3, 30, 34, 35, 48, 56). These data suggest that CDP may be involved in cellular differentiation. In this study, we demonstrate that CDP binds to the HPV-6 E6, E7, and E1 regulatory regions and negatively regulates these promoter activities in undifferentiated keratinocytes. When keratinocytes are induced to differentiate, the level of nuclear CDP and, correspondingly, DNA binding activity decreases concomitantly with increased promoter activity. Exogenously added CDP blocks this increased activity.

MATERIALS AND METHODS Plasmid constructions. HPV-6W50 was used as the parental plasmid. All nucleotide numbers refer to the HPV-6W50 genome, which contains a 94-bp insertion at nt 7350 and a deletion of nt 27 and 28 relative to HPV-6b (19). The E6 promoter was cloned into a luciferase (luc) vector as previously described (42) and renamed E6pluc. The E7 regulatory region (E7R-1) was amplified from the E6 translational start site to the E7 translational start site by PCR using the primers indicated in Table 1. The location of the synthesized fragments is shown in Fig. 1. Similarly, the E1 regulatory region (E1R-1) was amplified from the E7 translational start site to the E1 translational start site by PCR with the primers indicated in Table 1. The E7R-1- and E1R-1-amplified sequences were cloned into the luc vector between the PstI and HindIII sites, following excision of the E6 promoter from E6pluc with PstI and HindIII. For electrophoretic mobility shift assays (EMSAs), the fragments both upstream and downstream from either the E7 or E1 transcriptional initiation start site (29, 44, 49) were also amplified by PCR and cloned into either pUC19 or the luc vector. E7p was amplified from the E6 start codon to the E7 transcriptional start site, E7R-2 was amplified from the E7 transcriptional start site to 146 bp upstream of the E7 translational start site, and E7R-3 was amplified from that site to the E7 translational start site. E1p was cleaved from E1R-1 by digestion with PstI, which recognizes a site in the 59 E1R-1 PCR primer, and DraI, which is located 6 bp upstream of the E1 transcriptional start site, and then cloned into pUC19 cut with PstI and SmaI. E1R-2 was also cleaved from E1R-1 by digestion with DraI and HindIII (which has a recognition site in the 39E1R-1 PCR primer) and cloned into the blunt-ended PstI site and the HindIII site of the luc vector. The primers used in this study were

FIG. 1. Location of fragments used in functional assays and EMSAs. The top panel shows the three HPV-6W50 early promoter regulatory regions. The arrows indicate E6, E7, and E1 transcriptional initiation start sites. The dashed lines indicate E6, E7, and E1 translational start sites, respectively. The middle panel shows the three regulatory regions used in the functional studies. E6p extends from nt 7968 within the LCR to nt 96, 4 bp upstream of the E6 translational start site. E7R-1 extends from the E6 translational start site to the E7 translational start site; E1R-1 extends from the E7 translational start site to the E1 translational start site. In the bottom panel, the fragments used in EMSAs are shown. E7R-1 was divided into E7p, E7R-2, and E7R-3; E1R-1 was divided into E1p and E1R-2. Sequences used to amplify fragments are listed in Table 1. The cloning strategy is provided in Materials and Methods.

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either synthesized in the Biochemistry Biotechnology Facility (BBF) at Indiana University School of Medicine or purchased (Gibco/BRL). All recombinant sequences were confirmed by DNA sequencing by the BBF. Promoter subfragments used in EMSA competition assays. The E6 promoter was cleaved with DraI. The 47-bp 59 fragment (6a) contains nt 7968 to 20. The 76-bp 39 fragment (6b) contains nt 21 to 96. A 50-bp overlapping fragment (6m) was synthesized, containing nt 7990 to 45. Similarly, the E7 promoter was subdivided into a 7a fragment (123 bp) and a 7b fragment (45 bp) by PstI digestion; 7a contains nt 100 to 222, and 7b contains nt 223 to 267. The 7a fragment was further subdivided into 7c and 7d fragments by NsiI digestion. The 74-bp 7c fragment contains nt 100 to 173; the 49-bp 7d fragment contains nt 174 to 222. An overlapping 36-bp fragment (7m) was synthesized from nt 157 to 192. The E1 promoter was subdivided into four fragments: 1a and 1b by AccI digestion and 1c and 1d by AlwNI digestion. 1a contains nt 528 to 616 (89 bp), 1b contains nt 617 to 670 (54 bp), 1c contains nt 528 to 585 (58 bp), and 1d contains nt 586 to 670 (85 bp). A 51-bp E1m fragment which overlaps 1c and 1d was synthesized, containing nt 560 to 610. The fragments are depicted in Fig. 10. Cell culture and transfections. Human primary keratinocytes were prepared from newborn foreskins as described by Rheinwald (45). Briefly, keratinocytes from finely minced foreskins were seeded in three 10-cm plates per foreskin, each plate containing a feeder layer of mitomycin-treated 3T3-J2 fibroblasts in E medium containing 10% fetal calf serum (Hyclone) and 0.4 mg of hydrocortisone per ml, 0.1 nM cholera toxin, 5 mg of transferrin per ml, 2 nM 3,39-5-triodo-Lthyronine, 5 ng of epidermal growth factor per ml, and 13 antibiotic-antimycotic solution (100 U of penicillin/ml, 0.1 mg of streptomycin per ml, and 0.25 mg of amphotericin B per ml) (all supplements from Sigma) (42). At 80 to 90% confluence, the cultures were split 1:3 into keratinocyte serum-free medium (SFM; Gibco/BRL) plus 100 mM gentamicin. At 80 to 90% confluence, the cells were trypsinized and counted. For transfection experiments, 1.2 3 105 cells were plated per well into 12-well plates in SFM with 100 mM gentamicin. Sixteen to 18 h later, transfections were carried out with a total of 2.2 mg of DNA per well, which included the luc test plasmid (E6pluc, E7R-1luc, or E1R-1luc), the CDP expression plasmid (CMV [cytomegalovirus] CDP or MT [major adenovirus late promoter-tripartite leader] CDP) or empty vector containing only the regulatory region (CMV or MT), and the internal control plasmid CMVb-galactosidase (CMVb-gal), by using the Polybrene transfection procedure previously described (42). For nuclear extract preparation, 106 cells were plated per 10-cm dish in SFM plus 100 mM gentamicin, and cells were harvested 40 to 48 h later. For differentiation studies, keratinocytes grown in SFM were transfected as described above. Immediately following transfection, one set of cells was switched to Dulbecco’s modified Eagle’s medium (DMEM) (with 1.8 mM Ca21) plus 10% fetal calf serum. Nuclear extracts were made from cells incubated as monolayers for 36 h in SFM, harvested by scraping, and suspended in 1.5% methylcellulose (dissolved in 1 part Ham’s medium–3 parts DMEM containing 5% fetal calf serum). Luciferase and b-galactosidase assays. Keratinocyte extracts were prepared 40 to 48 h after transfection by using the lysis buffer and protocol from the Tropix Galacto-Light kit (Promega). The luciferase and b-galactosidase activities were assayed by using the reagents and protocol provided by the kit. All luciferase activities were standardized to the internal control activities. The standardized luciferase activity obtained for the regulatory region-luc parental plasmid in the presence of empty vector (CMV) was set to 1.0. In the keratinocyte differentiation experiments, the values obtained in the differentiation media with the empty vector or CDP expressing vector were compared to the value obtained with the regulatory region cotransfected with the empty vector and maintained in SFM. Nuclear extract preparation and EMSAs. Nuclear extracts from keratinocytes were prepared by the method of Dignam et al. (13), as modified by Lee et al. (33). Double-stranded oligonucleotides were cloned into pUC19 or luc vector, released by restriction enzyme digestion, and labeled with Klenow fragment (Gibco/BRL) by using [a-32P]dATP (Amersham). Radiolabeled oligonucleotides were resolved by 4.0% (8.0% for oligonucleotides smaller than 80 bp) polyacrylamide gel electrophoresis. The portion of the gel containing the target probe was excised and soaked overnight in 13 Tris-EDTA buffer (TE [pH 8.0]) in a 37°C water bath. The gel piece was sedimented, 20 mg of glycogen (Boehringer Mannheim) was added as a carrier to the supernatant, the oligonucleotide was precipitated with 100% ethanol, and the probe was recovered from the precipitated pellet. EMSAs were performed as described previously (42). Briefly, 3 to 6 mg of nuclear extract was mixed with 1.0 mg of poly(dI-dC) (Pharmacia) and competitor double-stranded oligonucleotides or anti-CDP antiserum or preimmune serum (a kind gift of Ellis Neufeld, Harvard Medical School), as indicated, in a 20-ml reaction volume containing 20 mM HEPES (pH 7.9), 150 mM KCl, 1 mM EDTA, 2 mM dithiothreitol, and 5% glycerol. Mixtures were incubated on ice for 15 min prior to the addition of 20,000 Cerenkov counts of 32P-labeled probe. After the addition of probe, the mixtures were incubated on ice for 10 min. The samples were then loaded onto a 0.53 TBE (Tris-borate-EDTA)–3.5% nondenaturing polyacrylamide gel, and electrophoresis was carried out at 280 V for 2.5 h at 4°C. Oligonucleotide CDP-a, which has a high affinity for CDP as previously described (48), was used as a positive control. An unrelated oligonucleotide, YY-1, was used as a negative control (42). Protein extraction and immunoblot analysis. Human foreskin keratinocytes were grown as submerged monolayers in SFM or in DMEM containing fetal calf

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FIG. 2. CDP negatively regulates the HPV-6 E6 promoter. E6pluc was cotransfected with either empty vector carrying the promoter (CMV or MT) or the CDP expression plasmid (CMVCDP or MTCDP) into human primary keratinocytes, along with the internal control CMVb-gal. Forty to 48 h later, the cells were harvested and the luciferase (Luc) activities were assayed as described in Materials and Methods. The standardized luciferase activity obtained for the E6pluc parental plasmid in the presence of empty vector (CMV or MT) was set to 1.0. The average 6 standard deviation for three experiments is shown.

serum or suspended in methylcellulose. At the indicated times, whole-cell extracts were made by lysing the cells with 23 lysis buffer (20% glycerol, 4% sodium dodecyl sulfate [SDS], 120 mM Tris-HCl [pH 6.8]); the recovered protein was then boiled for 10 min and stored at 280°C. Protein concentrations were determined using the Bio-Rad DC microplate assay. Fifty micrograms of protein was separated on 8% (for involucrin) or 15% (for keratin K10) polyacrylamide gels, transferred to nitrocellulose (Protan), and assayed by using the Bio-Rad Immun-Star chemiluminescent protein detection system. Primary antibodies were used at the following dilutions: keratin 10 (Zymed), 1:1,000; involucrin (Sigma), 1:1,000. Fifty micrograms of nuclear extract was analyzed for the presence of CDP by using a 1:2,000 dilution of anti-CDP antiserum. Goat anti-mouse antiserum (Bio-Rad) and goat anti-guinea pig antiserum (Sigma) were diluted 1:3,000. Nucleotide sequence accession number. The GenBank accession number for nt 7968 to 670 of HPV-6W50 is AF 126428.

RESULTS CDP binds the HPV-6 E6 promoter and negatively regulates its activity. In prior studies, we demonstrated that a 66-bp oligonucleotide, the 66-mer, located about 600-bp upstream of the E6 promoter, binds CDP and negatively regulates the HPV-6 E6 promoter activity (42). To determine whether CDP can also regulate E6 promoter activity without the involvement of the 66-mer, cotransfection experiments with the E6pluc plasmid and a CDP expression plasmid were conducted. CDP expressed from the CMV promoter or the MT promoter downregulated the E6 promoter activity severalfold in primary keratinocytes cultured in SFM (Fig. 2). Since CDP negatively regulated E6 promoter activity, EMSAs were used to determine whether CDP bound the E6 promoter (E6p). We first tested whether E6p would compete with the 66-mer for binding to CDP. The 66-mer was radiolabeled and mixed with keratinocyte nuclear extracts, which contain CDP DNA binding activity (42). As previously reported (42), the CDP complex was formed (C1 complex [Fig. 3, top panel, lane 2]). This complex was competed by unlabeled 66-mer (Fig. 3, [top panel, lanes 3 to 5]) and a CDP-a oligonucleotide, which is a strong CDP binder, (Fig. 3, top panel, lanes 9 to 11), but not by an unrelated, YY-1, oligonucleotide (Fig. 3, top panel, lanes 12 to 14). Interestingly, the C1 complex was also competed by the E6 promoter (Fig. 3, top panel, lanes 6 to 8), which suggests the E6 promoter may also bind CDP. To test this possibility, the E6 promoter was radiolabeled, and EMSAs were conducted with the keratinocyte nuclear extract. A similar C1 complex was formed with the E6 promoter (Fig. 3, bottom panel, lane 2). This complex was competed by unlabeled E6 promoter (Fig. 3, bottom panel, lanes 6 to 8), the

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FIG. 4. Confirmation of CDP binding to the E6 promoter by using anti-CDP antiserum. The 66-mer and the E6 promoter (E6p) probes were used in EMSAs. Lanes: 1 and 5, free probes (F); 2 and 6, assays performed in the absence of serum; 3 and 7, assays performed in the presence of anti-CDP antiserum (I); 4 and 8, assays performed in the presence of preimmune serum (PI).

FIG. 3. CDP binds to the HPV-6 E6 promoter in addition to the 66-mer. EMSAs were carried out with the 66-mer (upper panel) and the E6 promoter (lower panel) as probes. Lane 1 shows each free probe; lane 2 shows the result of the binding assay in the absence of competitors. The remaining lanes show competition assays in the presence of a 50-, 100-, or 500-fold molar excess of the indicated competitors.

66-mer (Fig. 3, bottom panel, lanes 3 to 5) and the CDP-a oligonucleotide (Fig. 3, bottom panel, lanes 9 to 11), but not by the YY-1 oligonucleotide (Fig. 3, bottom panel, lanes 12 to 14). These EMSA results strongly suggest CDP binds to the E6 promoter, resulting in the formation of the C1 complex. The CDP complex, formed by the E6 promoter probe, was completely disrupted by a 50-fold molar excess of CDP-a oligonucleotide, a 100-fold molar excess of the E6 promoter, and a 500-fold molar excess of the 66-mer (Fig. 3, bottom panel, lanes 9, 7, and 5, respectively). These data suggest that the CDP-a oligonucleotide has the highest CDP binding activity and that the E6 promoter has a higher affinity for CDP than does the 66-mer. The same relationship was seen when the 66-mer was used as the probe (Fig. 3, top panel). To confirm that CDP is present in the C1 complex formed with the E6 promoter, anti-CDP antiserum was included in the EMSAs. If the C1 complex contains CDP, addition of antiCDP antiserum may disrupt the complex or may result in a supershift of the C1 complex. Indeed, the C1 complex (Fig. 4, lane 6) was supershifted to the top of the gel by anti-CDP antiserum (Fig. 4, lane 7) but was unaffected by preimmune serum (Fig. 4, lane 8). Thus, CDP is present in the C1 complex formed by the E6 promoter. As a positive control, when the 66-mer was used as the probe, the C1 complex was similarly supershifted (Fig. 4, lanes 2 to 4), as reported previously (42). CDP negatively regulates the HPV-6 E7 and E1 promoter activities. In undifferentiated keratinocytes, transcription of high-risk HPV E7 initiates from the E6 promoter (49). In contrast, in the low-risk HPVs, there is an E7 promoter in addition to the E6 promoter which regulates E7 expression

(49). Since CDP binds and negatively regulates E6 promoter activity in HPV-6aW50, a low-risk HPV, it was of interest to determine whether the E7 promoter was regulated by CDP. In addition, since the E1 promoter is a differentiation-dependent promoter (26, 50), it was also a candidate for negative regulation by CDP in undifferentiated cells. Inspection of the E7 and E1 promoter sequences, as compared to the PCR-selected CDP binding sites (2, 5, 23), suggested that there were AT-rich sequences which CDP might bind in the E7 and E1 promoters. To position the luc gene in the approximate position of E7, the fragment from the E6 translational start site to the E7 translational start site was amplified for functional studies (Fig. 1 [E7R-1]). The same strategy was applied to amplify the E1 regulatory region and position the luc gene at the E1 translational start site (Fig. 1 [E1R-1]). Cotransfections were performed with plasmids encoding CDP expressed from the CMV promoter and E7R-1luc or E1R-1luc. CDP negatively regulated the activities of E7R-1 and E1R-1, as well as E6 promoter activity (Fig. 5). The activity of the internal control, CMVb-gal, only varied by 60.2 (data not shown). CDP binds the HPV-6 E7 and E1 promoters. To determine whether CDP binds to the E7 and E1 regulatory regions, the regions were subdivided into several fragments. E7R-1 was divided into the E7 promoter fragment (E7p) and E7R-2 and E7R-3 downstream fragments, and E1R-1 was divided into the E1 promoter fragment (E1p) and E1R-2 downstream fragment (Fig. 1). Radiolabeled E7p and E1p were used in EMSAs with nuclear extract from undifferentiated keratinocytes. C1 complexes were observed with both E7p and E1p probes (Fig. 6, top panel, lanes 2 and 10, respectively). The C1 complexes were competed by the CDP-a oligonucleotide (Fig. 6, top panel, lanes 3 to 5 and 11 to 13), but not by the YY-1 oligonucleotide (Fig. 6, top panel, lanes 6 to 8 and 14 to 16). These results suggested that CDP was a component of the C1 complexes. To confirm the presence of CDP in the C1 complexes, EMSAs with anti-CDP antiserum and preimmune serum were performed. Addition of anti-CDP antiserum but not preim-

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FIG. 5. CDP negatively regulates E6, E7, and E1 promoter activities. Cotransfection assays were carried out with E6luc, E7R-1luc, and E1R-1luc with either a plasmid carrying the CMV promoter (vector) or CMV-driven CDP (CDP) in human primary keratinocytes. The luciferase activity was normalized as for Fig. 2. The average 6 standard deviation for a representative experiment, conducted in duplicate, is shown.

mune serum caused a supershift of the C1 complexes (Fig. 6, bottom panel, compare lanes 7 and 11 to lanes 8 and 12, respectively), indicating that the E7 and E1 promoters, like the E6 promoter, bind CDP. The E7 promoter has the highest CDP binding affinity. To determine the relative affinity of CDP for these three promoters, each promoter was radiolabeled, and competition experiments were conducted with all three promoters as unlabeled competitors. The CDP complex formed by the E6 promoter (E6p) probe and keratinocyte nuclear extract was completely competed by a 20-fold molar excess of unlabeled E7 promoter (E7p) (Fig. 7). The E6 promoter-CDP complex was competed less well by the same amount of unlabeled E6 promoter and least well by the E1 promoter (E1p). This suggests that the E7 promoter has the highest CDP binding affinity, followed by the E6 promoter and then the E1 promoter. The same order was obtained with the E7 promoter (E7p) as a probe or the E1 promoter (E1p) as a probe and competition with the unlabeled promoters (Fig. 7). CDP binds fragments downstream of both the E7 and E1 transcriptional initiation start sites. Since the E7 and E1 regulatory regions used for functional assays contain sequences upstream and downstream of the transcriptional initiation start sites, the ability of CDP to bind the downstream fragments (E7R-2, E7R-3, and E1R-2) was tested. DNA-protein complexes that migrated at the position of C1 were observed when the radiolabeled downstream fragments were incubated with the keratinocyte nuclear extracts (Fig. 8). These complexes (Fig. 8, top panel, lanes 2 and 10; bottom panel, lane 2) were competed by the CDP-a oligonucleotide (Fig. 8, top panel, lanes 3 to 5 and 11 to 13; bottom panel, lanes 3 to 5), but not by the YY-1 oligonucleotide (Fig. 8, top panel, lanes 6 to 8 and 14 to 16; bottom panel, lanes 6 to 8). Similarly, the C1 complexes were supershifted by anti-CDP antiserum (Fig. 9, lanes 3, 7, and 11), but not by preimmune serum (Fig. 9, lanes 4, 8, and 12), indicating that those downstream fragments also bind CDP. The sizes of the E7 and E1 promoters, but not the E6 promoter, can be reduced with minimal loss of CDP binding activity. Multiple CDP binding sites have been reported within the gp91phox promoter (35) and the immunoglobulin heavy chain intronic enhancer (58). Furthermore, inspection of the E6, E7, and E1 promoter sequences (Fig. 10A) suggested that there were several candidate CDP binding sites (AT-rich re-

FIG. 6. CDP binds to the E7 and E1 promoters. (Top) EMSAs were performed with the E7 promoter (E7p) or the E1 promoter (E1p) as a probe. Lanes 1 and 9 show each free probe. Lanes 2 and 10 show assays in the absence of competitors (2). The remaining lanes show competition assays in the presence of a 20-, 50-, or 200-fold molar excess of the indicated oligonucleotide. (Bottom) The E6 promoter (E6p), the E7 promoter (E7p), and the E1 promoter (E1p) probes were used in EMSAs. Lanes 1, 5, and 9 show the free probes (F). The remaining lanes show assays performed in the absence of serum (2), in the presence of anti-CDP antiserum (I), or in the presence of preimmune serum (PI).

gions). To determine if there are one or several CDP binding sites within the E6 promoter, the promoter was subdivided into two parts, 6a and 6b (Fig. 10B). An oligonucleotide (6m) was also synthesized to overlap the cleavage site generating 6a and 6b. These three regions were used in EMSAs to compete with the radiolabeled E6 promoter for CDP complex formation. As shown in Fig. 10B, none of these three regions could compete

FIG. 7. CDP binding affinities for E6, E7, and E1 promoters. The E6 promoter (E6p), E7 promoter (E7p), and E1 promoter (E1p) were used as probes in EMSAs, in the absence (2) or presence of a 20-, 50-, or 200-fold molar excess of the indicated competitors.

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FIG. 9. Confirmation of CDP binding to the E7 and E1 downstream fragments by using anti-CDP antiserum. E7R-2, E7R-3, or E1R-2 was used as a probe in EMSAs in the absence of antiserum (2), in the presence of anti-CDP antiserum (I), or in the presence of preimmune serum (PI). F, free probes.

FIG. 8. CDP binds to the E7 and E1 downstream fragments. EMSAs were carried out with E7R-2, E7R-3, or E1R-2 as a probe. Lanes 1 and 9 in the top panel and lane 1 in the bottom panel show each free probe (F). Lanes 2 and 10 in the top panel and lane 2 in the bottom panel show assays in the absence of competitors. The remaining lanes show competition assays in the presence of a 20-, 50-, or 200-fold molar excess of the indicated oligonucleotide.

for CDP as efficiently as the entire E6 promoter, suggesting that the entire region is required for optimal CDP binding. A similar approach was used to dissect CDP binding sites within E7p and E1p. When two E7 promoter subfragments, 7a and 7b, were used in competition with radiolabeled E7p, most of the CDP binding activity was found to reside in 7a (Fig. 10C). 7a was further subdivided into 7c, 7d, and an overlapping 7m oligonucleotide (Fig. 10C). None of these regions could compete as well as 7a (Fig. 10C), suggesting that, within the E7 promoter, this 123-bp fragment exhibits near-maximal DNA binding activity. Subdivision of the E1 promoter into 1a and 1b indicated that most of the CDP binding activity resided in 1a (Fig. 10D). Cleavage of E1p into 1c and 1d indicated that both fragments could compete with radiolabeled E1p, but neither competed as well as the intact E1p. This was not due to cleavage within a critical CDP binding site, since the overlapping region, E1m, did not compete efficiently with E1p for CDP binding (Fig. 10D). Thus, within the E1 promoter, near-maximal CDP binding activity can be narrowed to the 89-bp 1a fragment. Increased HPV-6 early promoter activities coincide with the decreased CDP binding activity when keratinocytes are induced to differentiate. The HPV life cycle is tightly linked to keratinocyte differentiation. In undifferentiated keratinocytes,

HPV DNA is maintained at a low copy number, and viral gene expression is limited. When the keratinocytes differentiate, amplification of viral DNA and transcription is observed. In other cellular systems, CDP functions as a transcriptional repressor in undifferentiated cells, but not in differentiated cells. Thus, CDP is a cellular candidate for playing a role in the differentiation-dependent HPV life cycle. To test this hypothesis, undifferentiated keratinocytes were induced to differentiate by incubation in DMEM (high calcium) containing 10% fetal calf serum (4, 20, 28) or in semisolid medium (1.5% methylcellulose) (4, 46). Expression of the differentiation-dependent genes, coding for involucrin and keratin 10, increased when cells were suspended in methylcellulose (Fig. 11A). Similar results were obtained when cells were grown in DMEM containing 10% fetal calf serum (data not shown). By immunofluorescent staining, 100% of cells grown in methylcellulose for 48 h expressed involucrin, compared to 30% of cells grown in DMEM containing serum, and 1% of cells grown in SFM (data not shown). Keratinocytes plated in SFM were cotransfected with the CDP expression plasmid or the CMV empty vector and either the E6pluc, E7R-1luc, or E1R-1luc reporter plasmid. Transfected cells were induced to differentiate with high levels of calcium, because this method was more amenable to the analysis of multiple transfection variables within a given experiment. As shown in Fig. 12 (top panel), the luciferase expressed from all three regulatory regions in the presence of empty vector increased upon keratinocyte differentiation. Based on the immunofluorescence data, these cultures represent a mixed population of undifferentiated and differentiated cells, and, as such, may provide an underestimate of the extent of increased activity in a pure differentiated population. Less of an increase in promoter activity was detected in DMEM containing 10% fetal calf serum when the luc plasmids were cotransfected with the CDP expression plasmids. To determine whether the increased activities correlated with the loss of CDP, endogenous CDP levels were assayed and DNA binding activity was determined by using EMSAs with nuclear extracts from cells induced to differentiate in 1.5% methylcellulose. As shown in Fig. 11B, the amount of CDP was significantly decreased following induction of differ-

FIG. 11. Keratinocyte markers indicate cells suspended in methylcellulose are differentiated. (A) Fifty micrograms of whole-cell extract from cells suspended in methylcellulose was separated on polyacrylamide gels, and the proteins were transferred to nitrocellulose and probed with antibodies to involucrin (INV) or keratin 10 (K10). (B) Fifty micrograms of the nuclear extract used in EMSAs was similarly analyzed for the presence of CDP. S, Cells grown in SFM for the indicated time in hours (36 h in panel B); M, cells grown in 1.5% methylcellulose for the indicated time in hours.

entiation. In addition, after keratinocytes were suspended in methylcellulose, CDP DNA binding activity was greatly decreased (Fig. 12, bottom panel). Failure to detect CDP in nuclear extracts, by EMSA or by immunoblotting, was not due to the inability to isolate CDP from cells treated with methylcellulose, since functional CDP could be recovered after a short incubation (5 min) in methylcellulose (data not shown). Also, the quality and quantity of the extract were verified by Coomassie blue staining of an aliquot of a similar quantity of protein isolated from cells incubated for 48 h in SFM and methylcellulose and separated by SDS-polyacrylamide gel electrophoresis (data not shown). DISCUSSION We have previously shown that CDP binds to a 66-bp sequence located in the 59 end of the HPV-6W50 LCR (42). We have extended this finding and now demonstrate CDP binds to the E6, E7, and E1 promoters. The presence of CDP in the DNA-protein complexes was confirmed by two assays. As shown in Fig. 6 and 7, the CDP-a oligonucleotide, which binds with high affinity to CDP, competed with the E6, E7, and E1 promoters for C1 complex formation. In addition, anti-CDP antiserum supershifted the C1 complex. Cotransfection assays showed that CDP negatively regulated these three promoters in keratinocytes cultured in SFM (Fig. 2 and 5). Interestingly, when keratinocytes were induced to differentiate, the increased expression from the E6, E7, and E1 promoters correlated with a decrease in the quantity of nuclear CDP (and, correspondingly, of C1 complexes). These promoters were less active in the differentiated cells when a CDP expression plasmid was added to the transfections. The data strongly suggest that CDP is involved in control of the HPV-6 life cycle, which is dependent on keratinocyte differentiation. Based upon data obtained from monolayer cultures, the combinatorial effect of both negative regulators and positive regulators interacting with cis elements in the LCR most likely dictates the level of HPV gene expression. The HPV replication cycle is up-regulated during keratinocyte differentiation. During this differentiation, the expression of cellular transacting factors must change, which may either decrease the binding or activity of one or more negative regulatory proteins

FIG. 10. At least two regions within the E6, E7 and E1 promoters are required for CDP to bind. (A) Sequences of the E6, E7, and E1 promoters. Numbers indicate the nucleotide positions in the HPV-6aW50 genome. Underlined sequences indicate the restriction sites, and the corresponding restriction enzymes are listed above the recognition sequences. (B) E6 promoter subfragment competitors and EMSAs. (Top) The diagram depicts the relationship of the subfragments used in the EMSAs. The length of each fragment is marked within each fragment (see Materials and Methods for detailed information about each fragment). (Bottom) EMSAs were carried out with the E6 promoter as a probe in the presence of no competitor or a 20-, 50-, or 200-fold molar excess of the indicated competitors. (C) E7 promoter subfragment competitors and EMSAs. (Top) Diagram of the fragments used in the EMSAs. (Bottom) EMSAs were conducted as for panel B with the E7 promoter as a probe and E7p, 7a, 7b, 7c, 7d, and 7m as unlabeled competitors. (D) E1 promoter subfragment competitors and EMSAs. (Top) Diagram of the subfragments used in the EMSAs. (Bottom) EMSAs were conducted as for panel B with the E1 promoter as a probe and E1p, 1a, 1b, 1c, 1d, and 1m as unlabeled competitors.

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FIG. 12. Increased HPV-6 early promoter activities correlate with the decreased CDP binding activity in differentiated keratinocytes. (Top) Cotransfection experiments were performed as in Fig. 5. The luciferase activities obtained in the differentiation media with the empty vector or CDP expression vector were compared to luciferase activities obtained with the regulatory region cotransfected with the empty vector (CMV) and maintained in SFM. FCS, fetal calf serum. The results represent the average 6 standard deviation of two experiments each conducted in duplicate. (Bottom) EMSAs were performed with the E6 promoter (E6p), E7 promoter (E7p), or E1 promoter (E1p) as a probe and nuclear extracts from keratinocytes growing in SFM (S) or from keratinocytes suspended in methylcellulose for 24 h (M24) or 36 h (M36). F, free probes.

and/or increase the binding or activity of positive activators. Proteins known to interact with the HPV LCR have the potential to change during differentiation. Apt et al. (4) showed that the ratio of Sp1 to Sp3 changes during differentiation and that a high ratio, determined by EMSAs, correlated with upregulation of HPV-16 E6 promoter activity, as determined by functional assays. Thierry et al. (54) showed in undifferentiated human keratinocytes and cervical carcinoma cell lines that the cellular transactivator AP1, a Jun/Fos heterodimer, transactivated the HPV-18 LCR. More recently, Kyo et al. (32) showed that in HPV-31-positive keratinocytes, E6 and E7 expression detected by in situ hybridization correlated with the pattern of AP1 expression by immunohistochemical analyses. In the HPV-11 LCR, there are two AP1 sequences. When these AP1 sites were mutated, the increase in expression seen with the wild-type LCR in the suprabasal layers of keratinocytes grown on rafts was no longer seen (61). Other cellular factors that are involved in keratinocyte differentiation are the tissue-restricted POU domain transcription factors, Skn-1a/i (1). They are encoded by a single gene, are generated through alternative splicing, and are expressed primarily in the differentiated layers of

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the epidermis. Skn-1a specifically stimulates the E6 promoter in HPV-16- and HPV-18-positive keratinocytes, which suggests Skn-1 may be a molecular linker between HPV gene expression and epidermal differentiation (1, 60). Finally, the HPV-11 LCR is negatively regulated by C/EBPb. Changes in C/EBP family members during differentiation are postulated to relieve this negative regulation (57). This is the first report demonstrating that CDP represses several HPV promoters. Interestingly, increased HPV-6 promoter activities detected in keratinocytes induced to differentiate correlated with a decrease in nuclear CDP (Fig. 11). This strongly suggests that CDP plays a role in regulating HPV-6 gene transcription during keratinocyte differentiation. Our data are consistent with reports indicating that CDP and/or its DNA binding activity significantly decreases upon cell differentiation. For example, the induction of gp91phox gene expression during myeloid differentiation correlates with the loss of CDP DNA binding activity (48). Also, CDP, which negatively regulates expression of the immunoglobulin heavy chain, is detected in early pre-B cells but not mature B cells. In late pre-B cells, CDP is present but binds DNA poorly (58), suggesting CDP is posttranslationally modified. Indeed, CDP binding activity can be regulated by phosphorylation (11, 12). Our results indicate that the entire 123-bp E6 promoter, 123 bp of the E7 promoter, and 89 bp of the E1 promoter are sufficient for near-optimal CDP binding to each promoter. Further subdivision resulted in fragments with a significantly decreased ability to bind to CDP (Fig. 10). The data suggest that CDP requires two or more specific interactions over an extended region, no one of which is sufficient but some combination of which is necessary for high-affinity binding. CDP contains four DNA binding domains (Cut repeat 1, Cut repeat 2, Cut repeat 3, and the homeodomain) with broad and overlapping DNA binding specificities for AT-rich sequences (2, 5, 23). Because of this, it is difficult to recognize CDP binding sites by sequence inspection. However, several AT-rich sequences are present within the implicated CDP binding regions. The combination of EMSA results and sequence analysis suggests that the high-affinity binding to the regulatory regions is the result of cooperative binding of individual CDP binding domains to specific DNA sequences. Given that only one CDP complex was detected in EMSAs with the individual promoters, even with addition of more nuclear protein (data not shown), it appears that CDP is binding as a monomer. It has also been suggested that multiple DNA binding domains may bring together different regions of DNA (5). It is possible that in the context of the entire HPV-6 genome, CDP coordinately regulates HPV-6 gene expression from several promoters. It will be challenging to delineate the relationship between regulation of HPV-6 gene repression by CDP and the cooperative binding of multiple CDP binding sites. The concept of cooperativity is consistent with the CDP literature. The binding specificities of the different CDP DNA binding domains were determined by PCR-mediated random oligonucleotide selection using either glutathione S-transferase (GST) fusions with each DNA binding domain or immunoprecipitation from COS cells overexpressing CDP (2, 5, 23). While GST fusions, which can dimerize, could bind to the oligonucleotides, fusions to maltose binding protein (MBP), which cannot dimerize, could not bind DNA. However, an MBP-Cut repeat 3-homeodomain fusion could bind DNA (23). In addition, cooperative binding of Cut repeat II and the homeodomain resulting in formation of a ternary complex on an oligonucleotide has been reported (2). Within the gp91phox promoter, one high-affinity CDP binding site and at least three distal low-affinity CDP binding sites were observed (35). In at

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least one case (CDP-b), full binding activity required sequences that overlap a previously identified binding site (CDPa). Within the human tryptophan hydroxylase (hTPH) regulatory region, there are two footprints, FP-I and FP-II. Both regions of DNA must be present for CDP to form a complex detected by EMSA (53). These data strongly suggest that either the DNA binding domains within a single CDP molecule or those between CDP molecules bind cooperatively to DNA. CDP has been proposed to be a general repressor of gene transcription in undifferentiated cells, where it binds a number of gene promoters and represses gene transcription (3, 7, 17, 31, 34, 35, 48, 51, 52, 55). Two distinct modes of repression have been documented. On the one hand, CDP competes with an activator protein which binds to an overlapping site (7, 35, 36, 48, 51, 52). On the other hand, a carboxyl-terminal fragment of CDP functions as an active repression domain in a distance-independent manner (36). In addition, it has been recently proposed that CDP, a matrix attachment-associated region (MAR) binding protein, may block the association of chromatin with the nuclear matrix and thereby inhibit the initiation of transcription (6). Any or all of these mechanisms may function in keratinocytes. In summary, we showed here that the cellular transcription factor CDP binds HPV-6W50 E6, E7, and E1 promoters and down-regulates these promoter activities in undifferentiated keratinocytes. Upon keratinocyte differentiation, loss of CDP correlates with increased promoter activity. Thus, we propose that CDP plays a key role in the HPV life cycle. In HPVpositive keratinocytes, CDP inhibits HPV gene transcription in the basal layer, which contributes to the barely detectable viral gene transcription. Upon keratinocyte differentiation, CDP loses its inhibitory effect. With the loss of CDP (and perhaps other differentiation-dependent negative regulators) and the gain of differentiation-dependent transcriptional activators, the amplification of viral gene transcription is observed.

9.

10. 11. 12. 13. 14.

15. 16.

17. 18. 19. 20. 21. 22.

ACKNOWLEDGMENTS This work was supported by NIH grant AI31494. We thank Ellis Neufeld for providing the anti-CDP antiserum; Jean Bang and Grova Mae Lewis for excellent technical assistance; and Maureen Harrington, David Skalnik, Lucinda Carr, and Michael Klemsz for helpful discussions and critically reading the manuscript.

23. 24.

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