The promoter P7535 of human papillomavirus type 8 and the promoter P7185 of bovine papillomavirus type. 1 are negatively regulated by viral E2 proteins via ...
JOURNAL OF VIROLOGY, Oct. 1997, p. 8029–8034 0022-538X/97/$04.0010 Copyright © 1997, American Society for Microbiology
Vol. 71, No. 10
Competitive Binding of Viral E2 Protein and Mammalian CoreBinding Factor to Transcriptional Control Sequences of Human Papillomavirus Type 8 and Bovine Papillomavirus Type 1 HANNS-MARTIN SCHMIDT, GERTRUD STEGER,
AND
HERBERT PFISTER*
Institut fu ¨r Virologie der Universita ¨t zu Ko ¨ln, 50935 Cologne, Germany Received 10 April 1997/Accepted 8 July 1997
The promoter P7535 of human papillomavirus type 8 and the promoter P7185 of bovine papillomavirus type 1 are negatively regulated by viral E2 proteins via the promoter proximal binding sites P2 and BS1, respectively. Mutations of these E2 binding sites can reduce basal promoter activity. This suggests binding of a transcription-stimulating factor and may indicate that repression by E2 is due to competitive binding of viral and cellular proteins. A computer search revealed putative binding sites for core-binding factor (CBF; also referred to as PEA2, PEBP2, or AML), overlapping with P2 and BS1. Binding of recombinant CBF proteins to these sites was confirmed by band shift analysis. Competition of CBF and E2 protein for DNA binding was shown for both human papillomavirus type 8 and bovine papillomavirus type 1. The importance of CBF-E2 competition in E2-mediated repression could be demonstrated by comparing the E2 effect on P7185 activity in two cell lines containing different amounts of endogenous CBF. In cells with large amounts of CBF, E2 repressed P7185 wild-type constructs to the basal promoter activity of a mutant (50%) that could not bind this protein any more. In contrast, in a cell line containing small amounts of CBF, the promoter activities of constructs with wild-type and mutated CBF binding sites hardly differed and specific repression by E2 was not detectable.
for the polyomavirus enhancer binding protein 2 (PEBP2), the mouse CBF, overlaps with an E2 binding site that mediates repression (11). PEBP2 is a heterodimer, which is, like other CBFs, composed of a runt homology domain a subunit that can bind DNA and a b subunit that is unable to bind to the CBF site alone but enhances the binding of the a protein (19). At least three related mammalian genes code for CBFa subunits, but probably only one codes for CBFb (33, 34). Further complexity is achieved by differential transcription initiation, polyadenylation, and splicing of the CBF transcripts (3, 34). PEBP2 appears to be involved in transcriptional regulation of certain differentiation-related genes, e.g., osteoblast-specific genes (4). Its own expression seems to be linked to the differentiation state of cells, as PEBP2 became detectable only after differentiation induction in F9 mouse embryonal carcinoma cells (7). According to the CBF consensus binding site 59-Pu/TACC PuCA as described in reference 18, the P7185 promoter of BPV1 also contains a potential CBF site overlapping with the promoter proximal E2 binding site (see Fig. 1B). In order to test whether the theoretical CBF binding sites in HPV8 and BPV1 do indeed play a role in promoter regulation, we first performed binding assays with recombinant PEBP2 protein. PEBP2 binds to the CBF site in HPV8 and BPV1. Truncated PEBP2aA1 protein (containing the DNA binding and dimerization domains) was synthesized in Escherichia coli harboring pQEaN94C226 and purified as described elsewhere (11). PEBP2b protein was produced with a N-terminal His tag by inserting the 817-bp BamHI-NdeI fragment of pET3ab2 (19) between the BamHI and NdeI sites of pET14b (Novagen, Inc., Madison, Wis.). The recombinant protein was purified on a nickel-agarose column (Qiagen) as described by the manufacturer. Electrophoretic mobility shift assays with recombinant PEBP2 proteins were carried out in the presence of 10 mM Tris-Cl (pH 7.0), 100 mM KCl, 1 mM MgCl2, 0.005% poly(dIdC), 0.05% bovine serum albumin, 1 mM dithiothreitol 1 mM
The life cycle of papillomaviruses strongly depends on the differentiation state of the infected epithelial cell (6). Transcriptional control of early and late viral genes is thereby mediated by a complex interplay of viral and cellular factors, mainly binding to the noncoding regulatory sequences of papillomavirus DNA between the L1 and E6 genes. An increasing number of both positively and negatively acting host transcription factors has been identified, including YY1, SP1, AP1, Oct-1, and NF-1. Further modulation of transcriptional activity is achieved via the viral E2 proteins, which bind as dimers to the palindromic DNA sequence 59-ACCN6GGT, represented several times in the noncoding regulatory sequences of all papillomaviruses. The E2 proteins can act both as repressors and as activators (reviewed in reference 24). Repression could principally occur through diverse mechanisms, e.g., quenching, squelching, or competition for DNA binding (13). The promoter P7535 of human papillomavirus type 8 (HPV8) and the promoter P7185 of bovine papillomavirus type 1 (BPV1) are negatively regulated by viral E2 proteins via the promoter proximal binding sites P2 and BS1, respectively (27, 29, 32). The sequences around these binding sites are highly conserved among different papillomaviruses (28). These sites also seem to bind transcriptional activators, since mutations of these E2 binding sites not only abolish E2 binding but also lower the basal activity of the promoters. These data support the hypothesis that repression is due to competition between E2 and cellular factors. As noted before, the E2 binding site P2 of HPV8 overlaps with a theoretical core binding factor (CBF; also referred to as PEA2, PEBP2, or AML) binding site (14, 21) (see Fig. 1A). It has recently been demonstrated for BPV4 that a binding site
* Corresponding author. Mailing address: Institut fu ¨r Virologie der Universita¨t zu Ko ¨ln, Fu ¨rst-Pu ¨ckler-Strasse 56, 50935 Cologne, Germany. Phone: 49-221-478-4481. Fax: 49-221-407490. 8029
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FIG. 1. Recombinant PEBP2 protein recognizes the assumed binding sites close to the HPV8 P7535 and the BPV1 P7185. The sequences of the HPV8 and BPV1 oligonucleotides are shown beneath panels A and B, with mutated nucleotides given in lowercase letters. (A) 32P-labeled oligonucleotides (250 pg), representing the HPV8 sequence from nt 7486 to 7516 (P2TATA and its derivatives P2MT, P2C, and P2MTPEBP) were incubated with 5 ng of PEBP2a and/or 20 ng of PEBP2b as indicated. Complexes were separated by electrophoresis and visualized by autoradiography. The a/b complexes partly dissociated during the gel run. (B) 32P-labeled BPV1-BS1 (80 pg), representing the BPV1 sequence from nt 7198 to 7228, and mutant derivatives (BPV1-MT1 and BPV1-MT2) were incubated with 100 ng of PEBP2a and/or 100 ng of PEBP2b as indicated. Exposure to X-ray film was longer than in the experiments shown in panels A and C. (C) 32P-labeled P2TATA and BPV1-BS1 were incubated with PEBP2a and -b as in the experiment shown in panel A. (D) 32P-labeled PEA2 (250 pg) (59-GATCACTGACCGCAGCTG-39, representing nt 5120 to 5133 of the polyomavirus enhancer sequence with GATC added at the 59 end; PEBP2 binding site shown in bold) was incubated with a 200-fold excess of the indicated oligonucleotides and with PEBP2 protein as in the experiment shown in panel A.
EDTA, 5% glycerol, and cold competitor oligonucleotides when appropriate. The indicated amounts of 32P-labeled oligonucleotides were incubated with protein for 30 min at room temperature in a reaction volume of 20 ml, then loaded on 6% polyacrylamide gels buffered in either 13 Tris-glycine-EDTA (Fig. 1A and C and Fig. 2B and C) (buffer described in reference 11) or 0.53 Tris-borate-EDTA and run at 160 V for approximately 90 min. As shown in Fig. 1A, the recombinant PEBP2a/b heterodimer binds to the wild-type (wt) oligonucleotide P2TATA, comprising the E2 palindrome and the assumed CBF site of HPV8. An exchange of 2 nucleotides (nt) in the 59 moiety of the presumable CBF binding site, which led to a fivefold de-
crease in P7535 promoter activity (30), abolished PEBP2a/b binding to oligonucleotide P2MT. This correlation supports the hypothesis that CBF modulates the activity of the HPV8 promoter P7535. The heterodimer did not bind, either, to the oligonucleotide P2MTPEBP, with two mutated nucleotides at the 39 end of the CBF recognition sequence (Fig. 1A). The mutations in P2C outside the CBF consensus site had also significantly decreased the basal promoter activity (30). However, the oligonucleotide P2C was even more efficiently shifted by PEBP2 protein than P2TATA, pointing to another transcription-activating factor affected by these mutations. The specificity of the detected complexes and the relative affinities of the different oligonucleotides to PEBP2 were confirmed by competition experiments using a 200-fold excess of the indicated oligonucleotides and 32P-labeled oligonucleotide PEA2,
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FIG. 2. PEBP2 competes with E2 proteins of both HPV8 and BPV1 for DNA binding. (A) The 32P-labeled oligonucleotide P2TATA (25 pg), encoding the HPV8 wt sequence, was incubated with 100 ng of recombinant PEBP2a and 5, 1, 0.2, or 0.04 ml of HPV8 E2 protein extract as indicated. A double complex of bound PEBP2a is visible here due to the longer run and the use of a 10% polyacrylamide gel for better resolution. (B) The 32P-labeled oligonucleotide BPV1-BS1 (35 pg), encoding the BPV1 wt sequence, was incubated with 100 ng of recombinant PEBP2a and 30, 3, or 0.3 ng of BPV1 E2 D1-203 as indicated. (C) The 32P-labeled oligonucleotide PEBP2TRE (70 pg), with the otherwise overlapping E2 and PEBP2 binding sites set 7 bp apart from each other (see scheme below), was incubated with protein as in the experiment shown in panel B. The original PEBP2 binding site was changed from 59AACCACA to 59CACCACA in order to impede binding at this site. However, upon incubation with PEBP2a alone, a weak, slower-migrating band was still observed which probably represents a complex of two a-monomers bound to the oligonucleotide.
representing the bona fide PEBP2 binding site described within the polyomavirus enhancer (12) (Fig. 1D). Gel retardation experiments with oligonucleotide BPV-BS1, representing nt 7198 to 7228 of the wt BPV1 sequence, demonstrated binding of PEBP2. A lower affinity of the BPV1 binding site compared to HPV8 P2TATA was suggested by smaller amounts of shifted BPV1-BS1 (Fig. 1C) and weaker competition for PEBP2 binding to oligonucleotide PEA2 (Fig. 1D). To test if the substitution of 2 bp in the BPV1 context would result in loss of PEBP2 binding, analogous to the HPV8 oligonucleotide P2MT, we constructed oligonucleotide BPV1MT1 (see scheme in Fig. 1B). As expected, the oligonucleotide BPV1-MT1 neither bound to PEBP2 protein nor could compete for PEBP2 binding to PEA2 (Fig. 1B and D). To see whether the different CBF binding-site sequences of BPV1 and HPV8 account for the weaker binding of BPV1-BS1 to PEBP2, we created oligonucleotide BPV1-MT2, with the HPV8 CBF site in a BPV1 background. The apparent binding affinity of this sequence to PEBP2 was similar to that of the wt BPV1 sequence (Fig. 1B), suggesting that, at least in vitro, the binding affinity is not only determined by the defined consensus sequence but also influenced by the adjacent nucleotides (probably 59 to the PEBP2 site, since the HPV8 and BPV1 sequences are identical within 9 nt of the 39 flank [compare schemes in Fig. 1A and B]). PEBP2 and E2 compete for binding of P2 and BS1 in vitro. For DNA binding competition with PEBP2a, we used recombinant baculovirus-expressed HPV8 E2 protein as described before (29) and BPV1 E2 repressor protein that lacks amino acids 1 to 203 (E2D1-203) produced in yeast (25). The binding specificity of these proteins has been demonstrated before by band shift and/or footprint analysis. As shown in Fig. 2A, binding of HPV8 E2 to P2 and binding of PEBP2a to P2 are mutually exclusive. Increasing amounts of E2 partially displaced PEBP2a, and no double complexes were visible. The
same holds true for BPV1 E2 and PEBP2a binding to BS1 (Fig. 2B). To obtain a control for double complex formation, we constructed an oligonucleotide with the otherwise overlapping binding sites 7 bp apart. As expected, a new, slowly migrating band was observed when this oligonucleotide was incubated with both proteins, which is likely to represent the double complex (Fig. 2C). Presence of CBF in RTS3b and C33A cells. Functional assays of HPV8 promoter constructs were originally carried out with RTS3b cells (20, 30). To test for binding activity homologous to that of mouse PEBP2, nuclear extracts from these cells were prepared as described in reference 23. DNA binding reactions were carried out in the presence of 20 mM HEPES (pH 7.9), 135 mM KCl, 4 mM spermidine, 0.01% singlestranded DNA, 0.1 mM EDTA, 0.05% Nonidet P-40, 0.01% poly(dI-dC), 0.1% bovine serum albumin, 5% glycerol, and cold competitor oligonucleotides when appropriate. As shown in Fig. 3, nuclear extracts of RTS3b cells retarded the PEA2 oligonucleotide described above. Binding was competed for with a 100- to 200-fold excess of unlabeled oligonucleotides PEA2, P2TATA (data not shown), and P2C, but not with P2MT (Fig. 3A). Addition of a CBF-specific antiserum (raised against a peptide encompassing the 17 N-terminal amino acids of human AML-1 [16]) resulted in two supershifted complexes (Fig. 3B). Gel retardation assays with the labeled HPV8 oligonucleotides P2TATA and P2C, but not with the P2MT oligonucleotide, revealed a CBF-specific complex. As already described for the recombinant PEBP2 protein, P2C had a higher binding affinity for this complex than P2TATA. The specificity of the complexes was demonstrated by competition with unlabeled PEA2 oligonucleotide (data not shown). These results clearly demonstrate CBF binding activity in RTS3b cells. When screening several further cell lines, we found that C33A cells contain significantly smaller amounts of CBF specific binding activity than RTS3b cells (Fig. 3). Meyers et al.
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FIG. 3. (A) The cell line RTS3b contains large amounts, and the cell line C33A contains small amounts of PEBP2-homologous binding activity (CBF). Equal amounts of total protein (12.5 mg per reaction) from nuclear extracts were incubated with 330 pg of 32P-labeled oligonucleotide PEA2 (carrying the PEBP2/ CBF binding site). The specificity of the complex was demonstrated by competition with a 100-fold excess of unlabeled oligonucleotide P2C or P2MT. (B) Gel mobility shift assays of RTS3b and C33A cell lysates in the presence or absence of an antiserum against human AML1. After 12.5 of mg of total protein from nuclear extracts was incubated with or without 2 ml of anti-human AML-1 serum for 15 min at room temperature, 250 pg of 32P-labeled PEA2 was added and further incubated with the proteins for 15 min as indicated. A 4% polyacrylamide gel was used for better resolution of the complexes.
have also found only small amounts of CBF in C33A cells (17). We could not demonstrate a supershift with C33A extract, which might be due to limited sensitivity of the assay or to the presence of a variant of CBF protein which is not recognized by the antiserum. In view of the obvious differences in CBF specific binding activity between RTS3b and C33A cells, we decided to compare P7185 reporter constructs in these two cell lines with regard to their basal transcription level and their ability to be repressed by BPV1 E2 protein. Functional significance of E2-CBF competition in vivo. The 131-bp HincII-NarI fragment of BPV1 (nt 7146 to 7276) containing E2 BS1 and P7185 was cloned into the SmaI site of the promoterless plasmid pAluc (5) in front of the luciferase gene. The two double point mutations mt1 and mt2 (Fig. 1B) of the CBF binding site were introduced into this wt construct by PCR mutagenesis by standard methods (2). To confirm that the luciferase RNA is correctly initiated at P7185, we performed a primer extension analysis with RNA of transfected C33A cells. Transcription was initiated at the correct position in wt, mt1, and mt2 constructs (data not shown). As shown in Fig. 4A, elimination of the CBF site in pAlucMT1 (mt1) led to a considerable drop in luciferase expression compared to that in the wt construct when expression was tested in RTS3b cells. The mutant pAlucMT2 (mt2) was functionally indistinguishable from the wt construct, which is in line with mt2 and wt BS1 showing similar affinities to PEBP2 protein in the electrophoretic mobility shift assay. In C33A cells, luciferase expression was less affected by the destruction of the CBF site (Fig. 4A), which can be explained by weaker activation of the wt construct due to the lower CBF binding activity. The basal promoter activity of the P7185 wt construct in
FIG. 4. Specific repression of P7185 luciferase constructs by E2 is detectable only in RTS3b cells. (A) Transient transfection of RTS3b and C33A cells with 3 mg of the indicated luciferase constructs and 100 ng of the full-length BPV1 E2 expression vector pC59. (B) Transient transfection of both cell lines with the indicated luciferase constructs and increasing amounts of the BPV1 E2 repressor. Efficiency of transfection in both experiments was calibrated by cotransfecting plasmid pRSVbgal and measuring b-galactosidase activity in the cellular extracts.
RTS3b cells was indeed two- to threefold higher than that in C33A cells, as roughly estimated by comparing luciferase expression relative to b-galactosidase expression from pRSVbgal used as an internal transfection control (data not shown). Preliminary results from testing in C33A cells indicate that the P7535 promoter of HPV8 is also only marginally affected by the mutation in the CBF binding site. Cotransfection of pC59 (37), encoding the full-length BPV1 E2, repressed the luciferase expression of both the wt and mt2 constructs to levels comparable to those for mt1 in RTS3b cells. In C33A cells, full-length E2 only slightly repressed luciferase expression of all constructs. Since mt1 cannot bind E2 any more, we wondered whether this small effect might be due to squelching or to nonspecific E2 activation of the pRSVbgal internal transfection control. We therefore performed titration experiments with an expression vector for the E2 repressor protein that lacks amino acids 1 to 203 and is not able to activate pRSVbgal (23a). As shown in Fig. 4B, repression of the wt P7185 was seen only in RTS3b cells, not in C33A cells, although similar amounts of repressor were active in both cell lines, as confirmed by comparable repression of the control plasmid p4X21 (described in reference 26) (data not shown). The slight repression seen in C33A cells with full-length E2 is hereby confirmed to be non-
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specific and dependent on the E2 transactivation domain, whereas in RTS3b cells specific repression is demonstrated. Taken together, the difference in luciferase expression between the P7185 wt construct and the mutant derivative that cannot bind CBF any more correlated with the different amounts of CBF binding activity in the two cell lines. The degree of repression by E2 protein corresponded to the extent of CBF-dependent stimulation and did not lead to expression levels lower than those of a mutant that cannot bind CBF any more. These results underline the importance of CBF-E2 competition in E2-mediated repression. The CBFs are thus likely targets of E2 repression in at least three cases now, namely, in HPV8, BPV1 (this study), and BPV4 (11). This argues for a decisive role of those factors in the life cycle of papillomaviruses. Since the basal activity of the P7535 of HPV8 could also be lowered by the mutation P2C (29, 30), which shows no negative effect on CBF binding in vitro, another factor binding within the E2-specific palindrome seems to contribute to the basal promoter activity. This could imply synergistic effects with CBF as described for several DNA binding factors. It is known that CBF proteins are required for the tissuespecific activity of diverse enhancers and promoters, including T-cell receptor enhancers (8), the polyomavirus enhancer (12, 35), the murine leukemia virus enhancer (38), and the granulocyte-macrophage colony-stimulating factor promoters (31). Although the CBF binding site alone cannot direct tissuespecific transcription when attached to a heterologous promoter, it is assumed that CBF binding proteins act as enhancer or promoter organizers to recruit tissue-specific factors through physical interaction (9). In this context, it is interesting to note that the M33 element of HPV8 contains an ets-like motif (10) close to the CBF binding site. Synergistic activation of promoters by Ets-1 and CBF has been described previously (8, 36) and might represent one of the diverse possibilities for fine tuning the expression of viral proteins. Evidence is accumulating that cooperative binding of CBF and other factors could reflect a common theme in viral enhancers (36, 39). It is interesting that in certain cell lines, the mouse PEBP2 protein is differentially regulated upon UV radiation (22), although its role in the cellular response to DNA damage remains unclear. The role of CBF in the life cycle of papillomaviruses, and whether CBF is one of the factors related to differentiationdependent expression of papillomaviral proteins, is not yet known. The expression and/or modification patterns of CBF in normal human skin could offer hints on the significance of the CBF proteins for the papillomaviral life cycle. A straightforward approach will be the analysis of CBF-related papillomaviral gene expression in raft cultures (see, e.g., references 1 and 15). We thank Y. Ito for the release of plasmids pQEaN94C226 and pET3ab2, kindly provided by M. E. Jackson, and S. W. Hiebert for the kind donation of AML antisera. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 274/A8) and by the Ko ¨ln Fortune Program/Faculty of Medicine, University of Cologne. REFERENCES 1. Asselineau, D., B. A. Bernard, C. Bailly, M. Darmon, and M. Prunie´ras. 1986. Human epidermis reconstructed by culture: is it “normal”? J. Investig. Dermatol. 86:181–186. 2. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.). 1992. Short protocols in molecular biology. Greene Publishing and Wiley Interscience, New York, N.Y. 3. Bae, S. C., E. Ogawa, M. Maruyama, H. Oka, M. Satake, K. Shigesada, N. A. Jenkins, D. J. Gilbert, N. G. Copeland, and Y. Ito. 1994. PEBP2aB/mouse AML1 consists of multiple isoforms that possess different transactivation potentials. Mol. Cell. Biol. 14:3242–3252.
NOTES
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4. Banerjee, C., S. W. Hiebert, J. L. Stein, J. B. Lian, and G. S. Stein. 1996. An AML-1 consensus sequence binds an osteoblast-specific complex and transcriptionally activates the osteocalcin gene. Proc. Natl. Acad. Sci. USA 93: 4968–4973. 5. Dong, X.-P., F. Stubenrauch, E. Beyer-Finkler, and H. Pfister. 1994. Prevalence of deletions of YY1-binding sites in episomal HPV 16 DNA from cervical cancers. Int. J. Cancer 58:803–808. 6. Fuchs, P. G., and H. Pfister. 1997. Molecular biology of HPV and mechanisms of keratinocyte transformation, p. 15–46. In G. Gross and G. von Krogh (ed.), Human papillomavirus infections in dermatovenereology. CRC Press, Boca Raton, Fla. 7. Furukawa, K., Y. Yamaguchi, E. Ogawa, K. Shigesada, M. Satake, and Y. Ito. 1990. A ubiquitous repressor interacting with an F9 cell-specific silencer and its functional suppression by differentiated cell-specific positive factors. Cell Growth Differ. 1:135–147. 8. Giese, K., C. Kingsley, J. R. Kirshner, and R. Grosschedl. 1995. Assembly and function of a TCRa enhancer complex is dependent on LEF-1-induced DNA bending and multiple protein-protein interactions. Genes Dev. 9:995– 1008. 9. Hiebert, S. W., W. Sun, J. N. Davis, T. Golub, S. Shurtleff, A. Buijs, J. R. Downing, G. Grosveld, M. F. Roussel, D. G. Gilliland, N. Lenny, and S. Meyers. 1996. The t(12;21) translocation converts AML-1B from an activator to a repressor of transcription. Mol. Cell. Biol. 16:1349–1355. 10. Horn, S., H. Pfister, and P. G. Fuchs. 1993. Constitutive transcriptional activator of epidermodysplasia verruciformis-associated human papillomavirus 8. Virology 196:674–681. 11. Jackson, M. E., and M. S. Campo. 1995. Both viral E2 protein and the cellular factor PEBP2 regulate transcription via E2 consensus sites within the bovine papillomavirus type 4 long control region. J. Virol. 69:6038–6046. 12. Kamachi, Y., E. Ogawa, M. Asano, S. Ishida, Y. Murakami, M. Satake, Y. Ito, and K. Shigesada. 1990. Purification of a mouse nuclear factor that binds to both the A and B cores of the polyomavirus enhancer. J. Virol. 64:4808– 4819. 13. Levine, M., and J. C. Manley. 1989. Transcriptional repression of eukaryotic promoters. Cell 59:405–408. 14. Melnikova, I. N., B. E. Crute, S. Wang, and N. A. Speck. 1993. Sequence specificity of the core-binding factor. J. Virol. 67:2408–2411. 15. Meyers, C., M. G. Frattini, J. B. Hudson, and L. A. Laimins. 1992. Biosynthesis of human papillomavirus from a continuous cell line upon epithelial differentiation. Science 257:971–973. 16. Meyers, S., J. R. Downing, and S. W. Hiebert. 1993. Identification of AML-1 and the (8;21) translocation protein (AML-1/ETO) as sequence-specific DNA-binding proteins: the runt homology domain is required for DNA binding and protein-protein interactions. Mol. Cell. Biol. 13:6336–6345. 17. Meyers, S., N. Lenny, and S. W. Hiebert. 1995. The t(8;21) fusion protein interferes with AML-1B-dependent transcriptional activation. Mol. Cell. Biol. 15:1974–1982. 18. Ogawa, E., M. Maruyama, H. Kagoshima, M. Inuzuka, J. Lu, M. Satake, K. Shigesada, and Y. Ito. 1993. PEBP2/PEA2 represents a family of transcription factors homologous to the products of the Drosophila runt gene and the human AML1 gene. Proc. Natl. Acad. Sci. USA 90:6859–6863. 19. Ogawa, E., M. Inuzuka, M. Maruyama, M. Satake, M. Naito-Fujimoto, Y. Ito, and K. Shigesada. 1993. Molecular cloning and characterization of PEBP2b, the heterodimeric partner of a novel Drosophila runt-related DNA binding protein PEBP2a. Virology 194:314–331. 20. Purdie, K. J., C. J. Sexton, C. M. Proby, M. T. Glover, A. T. Williams, J. N. Stables, and I. M. Leigh. 1993. Malignant transformation of cutaneous lesions in renal allograft patients: a role for human papillomavirus? Cancer Res. 53:5328–5333. 21. Reh, H., and H. Pfister. 1990. Human papillomavirus type 8 contains cisactive positive and negative transcriptional control sequences. J. Gen. Virol. 71:2457–2462. 22. Rutberg, S. E., L. R. Dolan, and Z. Ronai. 1994. PEBP2—a modulator of polyoma DNA replication. DNA Cell Biol. 13:865–874. 23. Schreiber, E., P. Matthias, M. M. Muller, and W. Schaffner. 1989. Rapid detection of octamer binding proteins with “mini extracts” prepared from a small number of cells. Nucleic Acids Res. 17:6419. 23a.Steger, G. Unpublished data. 24. Steger, G., J. Ham, F. Thierry, and M. Yaniv. 1993. The E2 proteins of papillomaviruses as transcription and replication factors, p. 263–278. In W. Doerfler and P. Boehm (ed.), Virus strategies. VCH, Weinheim, Germany. 25. Steger, G., J. Ham, O. Lefebvre, and M. Yaniv. 1995. The bovine papillomavirus 1 E2 protein contains two activation domains: one that interacts with TBP and another that functions after TBP binding. EMBO J. 14:329–340. 26. Steger, G., and S. Corbach. 1997. Dose-dependent regulation of the early promoter of human papillomavirus type 18 by the viral E2 protein. J. Virol. 71:50–58. 27. Stenlund, A., and M. R. Botchan. 1990. The E2 trans-activator can act as a repressor by interfering with a cellular transcription factor. Genes Dev. 4:123–136. 28. Stubenrauch, F., J. Malejczyk, P. Fuchs, and H. Pfister. 1992. Late promoter of human papillomavirus type 8 and its regulation. J. Virol. 66:3485–3493.
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29. Stubenrauch, F., and H. Pfister. 1994. Low-affinity E2-binding site mediates downmodulation of E2 transactivation of the human papillomavirus type 8 late promoter. J. Virol. 68:6959–6966. 30. Stubenrauch, F., I. M. Leigh, and H. Pfister. 1996. E2 represses the late gene promoter of human papillomavirus type 8 at high concentrations by interfering with cellular factors. J. Virol. 70:119–126. 31. Takahashi, A., M. Satake, Y. Yamaguchi-Iwai, S.-C. Bae, J. Lu, M. Maruyama, Y. W. Zhang, H. Oka, N. Arai, K. Arai, and Y. Ito. 1995. Positive and negative regulation of granulocyte-macrophage colony-stimulating factor promoter activity by AML1-related transcription factor, PEBP2. Blood 86: 607–616. 32. Vande Pol, S. B., and P. M. Howley. 1990. A bovine papillomavirus constitutive enhancer is negatively regulated by the E2 repressor through competitive binding for a cellular factor. J. Virol. 64:5420–5429. 33. Wang, Q., T. Stacy, J. D. Miller, A. F. Lewis, T.-L. Gu, X. Huang, J. H. Bushweller, J.-C. Bories, F. W. Alt, G. Ryan, P. P. Liu, A. Wynshaw-Boris, M. Binder, M. Marin-Padilla, A. H. Sharpe, and N. A. Speck. 1996. The CBFb subunit is essential for CBFa2 (AML1) function in vivo. Cell 87:697–708. 34. Wang, S., Q. Wang, B. E. Crute, I. N. Melnikova, S. R. Keller, and N. A.
J. VIROL.
35.
36. 37. 38. 39.
Speck. 1993. Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor. Mol. Cell. Biol. 13:3324–3339. Wasylyk, B., J. L. Imler, B. Chatton, C. Schatz, and C. Wasylyk. 1988. Negative and positive factors determine the activity of the polyoma virus enhancer a domain in undifferentiated and differentiated cell types. Proc. Natl. Acad. Sci. USA 85:7952–7956. Wotton, D., J. Ghysdael, S. Wang, N. A. Speck, and M. J. Owen. 1994. Cooperative binding of Ets-1 and core binding factor to DNA. Mol. Cell. Biol. 14:840–850. Yang, Y.-C., H. Okayama, and P. M. Howley. 1985. Bovine papillomavirus contains multiple transforming genes. Proc. Natl. Acad. Sci. USA 82:1030– 1034. Zaiman, A. L., A. F. Lewis, B. E. Crute, N. A. Speck, and J. Lenz. 1995. Transcriptional activity of core binding factor a (AML1) and b subunits on murine leukemia virus enhancer cores. J. Virol. 69:2898–2906. Zaiman, A. L., and J. Lenz. 1996. Transcriptional activation of a retrovirus enhancer by CBF (AML1) requires a second factor: evidence for cooperativity with c-myb. J. Virol. 70:5618–5629.
