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J. Biol. Chem., Vol. 277, Issue 47, 45619-45629, November 22, 2002

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Transcriptional Activity among High and Low Risk Human Papillomavirus E2 Proteins Correlates with E2 DNA Binding^*

Samuel Y. Hou, Shwu-Yuan Wu, and Cheng-Ming Chiang

From the Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4935

Received for publication, July 9, 2002, and in revised form, August 28, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The full-length E2 protein, encoded by human papillomaviruses (HPVs), is a sequence-specific transcription factor found in all HPVs, including cancer-causing high risk HPV types 16 and 18 and wart-inducing low risk HPV types 6 and 11. To investigate whether E2 proteins encoded by high risk HPVs may function differentially from E2 proteins encoded by low risk HPVs and animal papillomaviruses, we conducted comparative DNA-binding and transcription studies using electrophoretic mobility shift assays and cell-free transcription systems reconstituted with purified general transcription factors, cofactor, RNA polymerase II, and with E2 proteins encoded by HPV-16, HPV-18, HPV-11, and bovine papillomavirus type 1 (BPV-1). We found that although different types of E2 proteins all exhibited transactivation and repression activities, depending on the sequence context of the E2-binding sites, HPV-16 E2 shows stronger transcription activity and greater DNA-binding affinity than those displayed by the other E2 proteins. Surprisingly, HPV-18 E2 behaves more similarly to BPV-1 E2 than HPV-16 E2 in its functional properties. Our studies thus categorize HPV-18 E2 and BPV-1 E2 in the same protein family, a finding consistent with the available E2 structural data that separate the closely related HPV-16 and HPV-18 E2 proteins but classify together the more divergent BPV-1 and HPV-18 E2 proteins.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Human papillomaviruses (HPVs)¹ are a family of small DNA viruses that cause a wide variety of human diseases ranging from benign epithelial lesions, such as warts, to invasive cancers, such as cervical carcinoma. So far, more than 100 HPV types have been identified and fully sequenced, whereas more than 120 putative novel types have been partially characterized (1, 2). HPV types frequently found in invasive cancers include HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -58, and -59. These are classified as high risk HPVs. In contrast, HPV types that are rarely found in cancers but are associated with genital warts, such as HPV-6 and HPV-11, are considered low risk HPVs (1-3). Because the genomic structures of HPVs are highly conserved, it is important to determine the functional differences among individual HPV gene products which lead to etiologically high and low risk phenotypes. Previous comparative studies have primarily focused on HPV-encoded E6- and E7-transforming proteins. These studies found that E6 and E7, from high risk HPVs, lead to cellular transformation much more readily than low risk E6 and E7 proteins (for review, see Refs. 4 and 5). In both high and low risk HPVs, expression of E6 and E7 is transcriptionally regulated via the E6 promoter by many cellular and viral proteins. The full-length viral E2 protein is a sequence-specific transcription factor that functions as an activator or repressor to regulate tightly the E6 promoter through four consensus E2-binding sites (E2-BSs), ACCGN₄CGGT (6, 7), whose locations within the upstream regulatory region (URR) are highly conserved among genital HPVs. Efficient activation of the E6 promoter requires binding of E2 protein to the promoter-distal E2-BSs in conjunction with binding of cellular factors to the enhancer elements also located within the URR (8-11). Activation directly mediated through E2 protein may occur via different mechanisms. First, E2 may recruit the general transcription machinery to the promoter through direct interactions with TATA-binding protein (TBP), TBP-associated factors in TFIID, TFIIB, as well as RNA polymerase II (12-17). Second, E2 interacts with nuclear factors Sp1, Gps2/AMF-1, or TopBP1, which may then serve as coregulators for transcription (18-20). Third, E2 may potentiate transcription by remodeling DNA structure at the chromatin level (21). Indeed, studies have shown E2 interacts with proteins (CREB-binding protein, p300, and p300/CBP-associated factor) possessing histone acetyltransferase activity (22-24). It is likely that different types of E2 proteins function differentially in regulating preinitiation complex assembly and show varied cooperativity with cellular enhancer-binding factors that recognize constitutive, inducible, or cell type-specific enhancer elements located in the URR, as well as with general or gene-specific transcription cofactors that modulate E2 activity on HPV chromatin.

Although low levels of E2 occupy promoter-distal binding sites, it is postulated that transcriptional repression occurs with high levels of E2 leading to occupancy at promoter-proximal E2-BSs thus displacing cellular factors critical for E6 promoter activity (9, 10, 25-27). We have shown previously that E2 protein can also actively repress transcription by preventing preinitiation complex formation (28). This active repression was demonstrated to be independent of cellular enhancer-binding factors, Sp1, and general cofactors such as TBP-associated factors, TFIIA, mediator and PC4 (28). Conversely, cellular proteins may bind to DNA and hinder access of E2 protein, thus inhibiting E2 function (29-31). Papillomaviruses may also use its own proteins to modulate E2 activation function. These include various forms of E2 repressor proteins created through alternative splicing to generate gene products that contain the same DNA-binding domain linked to different N-terminal regions (32-34) or the use of viral E1 protein to abrogate E2 transactivation function through direct protein-protein interaction (35, 36). Because E2 only weakly activates (~2-fold) but strongly represses (up to 100-fold) the native HPV promoter in both transfected cells (9) and in currently available cell-free transcription systems (for review, see Ref. 28), our initial effort was focused on dissecting the mechanisms of transcriptional repression by various E2 proteins on their homologous E6 promoter (28).

Interaction between viral E1 and E2 proteins also regulates papillomavirus DNA replication and viral episome maintenance (37-40). In addition to binding to viral DNA, E2 ensures proper segregation of viral genomes during cellular replication which is independent of the E2 DNA-binding domain (41-43). Analogous to abrogation of E2 transactivation function, it has been suggested that E1 also regulates E2 binding to mitotic chromosomes (44). Besides regulating viral transcription, replication, and viral episome maintenance, E2 has been implicated in several cellular processes relevant to carcinogenesis. E2 transfected into HPV DNA-containing cell lines can lead to growth arrest, apoptosis, or abrogation of mitotic checkpoints (45-47). E2 repression of E6 and E7 oncogene expression leading to reactivation of p53 and pRb tumor suppressor pathways has been the proposed mechanism for growth arrest in HPV-positive cell lines (48, 49). Surprisingly, growth arrest and repression of E6 and E7 expression require an intact E2 transactivation domain (50-52). Expression of E2 protein in a transformed cell line devoid of HPV DNA also displayed growth arrest through an undefined mechanism (45, 51). E2-triggered apoptosis can occur through both p53-independent (53) and p53-dependent pathways (54). Similar to E2-induced growth arrest, E2 caused apoptosis in HPV-negative cell lines through unknown pathways (54).

Although many functions have been attributed to papillomaviral E2 proteins, very few studies have been conducted to compare directly the transcription activities among HPV and BPV-1 E2 proteins (11, 55, 56). Experiments directly comparing high risk HPV-16 and HPV-18 E2 proteins with low risk HPV-6 E2 protein by transient transfection assays found that high risk E2 proteins have higher activation activities than low risk E2 protein, but this activity did not correlate with either higher steady-state levels of protein in vivo or with DNA-binding properties of the two classes of E2 proteins (56). Therefore, it was hypothesized that high risk E2 proteins were intrinsically better transcriptional activators than low risk E2 proteins, which in turn regulate the levels of E6 and E7 oncoproteins in transformed cells and thereby control the processes leading to carcinogenesis. In a more recent study, high risk E2 was found to have higher affinity for transcriptional coactivator CREB-binding protein than that of low risk E2 (23). These studies on E2 transcriptional activity were assayed primarily in transiently transfected cells. Therefore, interpretation of the results may be complicated by interference of other cellular factors, potentially masking the true activity of E2 protein.

To address the differences in transcriptional activities between high and low risk HPV E2 proteins, we employed in vitro transcription systems reconstituted either with individually purified recombinant general transcription factors (TFIIB, TFIIE, and TFIIF), epitope-tagged protein complexes (TFIID, TFIIH, and pol II) and general cofactor PC4, or with a preassembled RNA polymerase II (pol II) holoenzyme complex and TBP (17, 28). Using this highly purified in vitro transcription system along with an HPV responsive G-less cassette template containing two E2-BSs (17), high risk HPV-16 E2 protein was found to possess greater transactivation activity than those exhibited by E2 proteins encoded by HPV-18, HPV-11, and BPV-1. For repression assays, we employed an in vitro transcription system reconstituted with pol II holoenzyme and TBP with HPV template containing either homologous or heterologous URR linked to a G-less cassette. HPV-16 E2 (16E2) was determined to be the strongest transcriptional repressor, followed by BPV-1 E2 (BE2), HPV-18 E2 (18E2) and HPV-11 E2 (11E2). The transcriptional activities of these E2 proteins correlated well with their corresponding binding affinities to E2-BSs derived from the promoter-proximal regions of naturally occurring HPV-11, HPV-16, and HPV 18 E6 promoters, as well as BPV-1. Although previous in vivo studies revealed little correlation between E2 transcriptional activities and DNA-binding affinities, our in vitro studies indicate that higher transcriptional activation and repression activities of various E2 proteins correlate directly with their relative DNA-binding affinities for E2-BSs.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasmid Constructions-- Bacterial expression plasmids pF:E2-11d (28), pF:16E2-11d, pF:18E2-11d, and p:BE2-11d were used to express HPV-11, HPV-16, HPV-18, and BPV-1 E2 proteins, respectively. The plasmid pF:16E2-11d was constructed by cloning the HPV-16 E2 open reading frame, generated by PCR amplification using pCMV-16E2 (56) as template with an NdeI site-containing sense primer (5'-AAGTCGACATATGGAGACTCTTTGCCAA-3') and a BamHI site-containing antisense primer (5'-AACTCGAGGATCCTCATATAGACATAAATCC-3'), into pF:E4-11d (28), after swapping the inserts between NdeI and BamHI sites. The plasmid pF:18E2-11d was created similarly by using pCMV-18E2 (56) as template with sense primer (5'-AAGTCGACATATGCAGACACCGAAGGAAA-3') and antisense primer (5'-AACTCGAGGATCCTTACATTGTCATGTATCCC-3') for PCR amplification. Likewise, the plasmid pF:BE2-11d was cloned using pdBPV-1(142-6) (57) as template with sense primer (5'-AAGTCGACATATGGAGACAGCATGCGAA-3') and antisense primer (5'-TTCTCGAGGATCCTATTGATGCAAGC-3').

The G-less cassette template p18URR-GLess, containing the HPV-18 URR spanning nucleotides 6929-7857/1-81, used for in vitro transcription was constructed by cloning the PCR product, generated from genomic HPV-18 DNA (gift from L. T. Chow) using an EcoRI site-containing sense primer (5'-CTGGGTACCGAATTCGGATCCCTATGATAAG-3') and an SacI site-containing antisense primer (5'-CTGAGATCTGAGCTCTTTTATATACACCG-3') into the EcoRI-SacI-linearized G-less cassette vector pGL (58). The p16URR-GLess G-less cassette template, spanning HPV-16 URR nucleotides 7007-7904/1-72, was constructed similarly by cloning the PCR product generated from genomic HPV-16 DNA (gift from P. M. Howley) using sense primer (5'-CTGGGTACCGAATTCAGACCTAGATCAGTTTC-3') and antisense primer (5'-CTGAGATCTGAGCTCTTTTATACTAACCG-3') into pGL. The other transcription templates, p11URR-GLess (originally named p7072-70GLess/I, see Ref. 28), p7862-70(23M)GLess/I, pML53, p2E2(IR)53, and pG₅MLT, have already been described (17, 28).

Protein Purification-- Bacterially expressed FLAG-tagged HPV-11 E2, HPV-16 E2, HPV-18 E2, and BPV-1 E2 were purified from BL21(DE3)pLysS strain harboring pF:E2-11d, pF:16E2-11d, pF:18E2-11d, and pF:BE2-11d, respectively, according to the protocol published previously (17, 28). Isolation of recombinant FLAG-tagged human general transcription factors (TFIIB, TBP, TFIIE, and TFIIE), FLAG-tagged multiprotein complexes (TFIID, TFIIH, and pol II), six histidine-tagged TFIIF subunits (RAP30 and RAP74), recombinant PC4, and TFIID-deficient pol II holoenzyme was conducted as described previously (17, 28, 59-61).

In Vitro Transcription-- The transcription repression assays were performed in a two-component transcription system reconstituted with TFIID-deficient pol II holoenzyme and TBP using 50 ng of p11URR-GLess, p16URR-GLess, p18URR-GLess, or p7862-70(23M)GLess/I G-less cassette template, 50 ng of pML53, 1 ng of TBP, and 3 µl (~ 90 ng) of pol II holoenzyme, in the absence or presence of 11E2, 16E2, 18E2, or BE2 protein as specified in the figures. Reactions were then conducted and analyzed as described previously (28, 60).

The E2 transactivation assay was carried out in a highly purified in vitro transcription system in a 30-µl reaction containing 50 ng of pG₅MLT, 50 ng of p2E2(IR)53, 10 ng of TFIIB, 20 ng of TFIID, 2.5 ng each of TFIIE and TFIIE, 2 ng of TFIIF, 22.5 ng of TFIIH, and 5 ng of pol II, in the presence or absence of 150 ng of PC4 and an increasing amount of 11E2, 16E2, 18E2, or BE2 as specified, following the two-step incubation protocol (17). Reactions were then performed and analyzed as described previously (17, 60). Transcription signals were quantified by PhosphorImager analysis (Molecular Dynamics).

Electrophoretic Mobility Shift Assay (EMSA)-- DNA probes used for EMSA were generated by PCR amplification of respective HPV E6 promoter-proximal fragments and BPV-1 URR. The HPV-11 E6 promoter-proximal fragments, HPV-11(7902-92), HPV-11(7902-92)3M, HPV-11(7902-92)4M, and HPV-11(7902-92)34M, which span HPV-11 nucleotides 7902-7933/1-92 containing wild-type 3 and 4, mutated 3, mutated 4, or mutated 3 and 4 E2-BSs, were created by PCR amplification using sense primer (5'-AACCCGGGTACCTACCCACACCCTACATA-3') and antisense primer (5'-GGACACAGATCTGAGCTCTGCTAATTTTTTGGG-3') with DNA templates pGL7072-161, p24-N-3M, p24-N-4M, and p24-N-34M (9, 28), respectively. The wild-type HPV-16 E6 promoter-proximal DNA fragment HPV-16(7868-96), spanning HPV-16 nucleotides 7868-7904/1-96 and containing two wild-type E2-BSs, was generated by PCR amplification of HPV-16 genomic DNA (gift from P. M. Howley) using sense primer (5'-CTGGGTACCGAATTCTTACACATTTACAAGCA-3') and antisense primer (5'-GGACACAGATCTGAGCTCTTTTGGTGCATAAA-3'). The wild-type HPV-18 E6 promoter-proximal DNA fragment HPV-18(7834-101), spanning HPV-18 nucleotides 7834-7857/1-101 and containing two wild-type E2-BSs, was generated by PCR amplification of HPV-18 genomic DNA (gift from L. T. Chow) using sense primer (5'-CTGGGTACCGAATTCTGGGCAGCACATACTAT-3') and antisense primer (5'-GGACACAGATCTGAGCTCATTGTGGTGTGTTTCTC-3'). The BPV-1 DNA fragment BPV-1(7874-85), spanning BPV-1 nucleotides 7874-7945/1-85 (numbering based on GenBank accession number NC_001522) and containing two wild-type E2-BSs, was generated by PCR amplification using pdBPV-1(142-6) as template (57) with sense primer (5'-CTGGGTACCGAATTCGCAGCATTATATTTTAAG-3') and antisense primer (5'-GGACACAGATCTGAGCTCAACCGGGGTCTGTCAGC-3'). These PCR-amplified DNA fragments were then purified by gel electrophoresis using a 2% agarose gel and used for end-labeling reactions with T4 polynucleotide kinase and [-³²P]ATP. DNA probes were purified further by NICK column (Amersham Biosciences).

EMSA was conducted in a 10-µl reaction containing ~0.5 fmol of ³²P-labeled DNA probe in the presence of 10 mM HEPES-Na (pH 7.9), 5 mM dithiothreitol, 0.2 mM EDTA, 4 mM MgCl₂, 10% glycerol, 0.1 mg/ml bovine serum albumin, 10 ng/µl poly(dI-dC), and increasing amounts of respective purified E2 proteins (adjusted to 2 µl in BC300). Reactions were incubated at 30 °C for 1 h and then loaded onto a 5% polyacrylamide gel in 0.5× TBE and run at 175 V for ~2.5 h at 4 °C. The gels were then dried and exposed to x-ray films. The signals were quantified by PhosphorImager analysis.

Calculation of Equilibrium Binding Constants-- The intensity of the remaining probe in each lane after EMSA was quantified using ImageQuant (Molecular Dynamics) without () or with ( for each lane) an increasing amount of 11E2, 16E2, 18E2, or BE2 protein. Fractional occupancy () was then determined by Equation 1.

(Eq. 1)

Binding isotherms were obtained by plotting fractional occupancy as a function of protein concentrations and analyzed by nonlinear least squares analyses. The equilibrium binding constant, K_d, was determined by analysis of the titration curves against Equation 2,

(Eq. 2)

where  = fractional occupancy, [protein] = protein concentration, and K_d = the equilibrium binding constant (62).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Transcriptional Repression Mediated by High and Low Risk HPV and BPV-1 E2 Proteins-- To compare the transcriptional activities between high risk (HPV-16 and HPV-18) and low risk (HPV-11) HPV and animal papillomavirus E2 proteins, we first expressed full-length HPV-11, HPV-16, HPV-18, and BPV-1 E2 proteins in bacteria. All E2 proteins share similar structural features with an N-terminal activation domain linked to a C-terminal DNA-binding/dimerization domain by a flexible hinge region (Fig. 1A). These bacterially expressed E2 proteins were purified to near homogeneity and clearly migrated at positions corresponding to their predicted molecular sizes (Fig. 1B). The amounts of various purified E2 proteins were then normalized by Western blotting with anti-FLAG monoclonal antibody, which recognizes the same epitope sequence introduced at the N terminus of individual E2 proteins (data not shown). For functional studies, we also created three transcription templates, containing enhancer elements and the E6 promoter derived from each type of HPV, by cloning respective URR into a G-less cassette of 377 nucleotides (Fig. 1C). We demonstrated previously that correct initiation from the HPV E6 promoter whose transcription is regulated by different amounts of E2 proteins, similar to the in vivo observations, was recapitulated in our cell-free transcription systems using the G-less cassette templates (28).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Comparison of HPV and BPV-1 E2 proteins and the sequences of different E2-BSs. A, structural features of E2 proteins. The N-terminal activation domain (AD) is linked to the C-terminal DNA binding/dimerization domain (DBD) of each E2 protein via the hinge (H) region. Numbers refer to boundaries of amino acids depicting the protein domains found in 11E2, 16E2, 18E2, and BE2 proteins, based upon reports published previously (72-75). The apparent molecular mass (in kDa) of each FLAG-tagged E2 protein, estimated by SDS-PAGE, is listed on the right. B, Coomassie Blue-stained gel of purified E2 proteins. Recombinant FLAG-tagged BE2 (lane 1), 11E2 (lane 2), 16E2 (lane 3), and 18E2 (lane 4) were expressed in and purified from bacteria, resolved by 10% SDS-PAGE, and visualized after Coomassie Blue staining. Positions of prestained protein size markers (in kDa) are indicated on the left. C, transcriptional templates containing natural HPV URR used for in vitro transcription assays. Constructions of various G-less cassettes driven by HPV-11 URR (p11URR-GLess), HPV-16 URR (p16URR-GLess), or HPV-18 URR (p18URR-GLess) were as described under "Experimental Procedures." Each template contains the naturally occurring HPV URR and the TATA box of the E6 promoter preceding a 377-nucleotide G-less cassette. Nucleotide numbering for boundaries of E2-BSs is based upon previous studies (9, 28) and HPV nucleotide accession numbers AF125673 (for HPV-16) and X05015 (for HPV-18).

When examined in a cell-free transcription system reconstituted with TBP and human pol II holoenzyme, which provides pol II and general transcription factors TFIIB, TFIIE, TFIIF, and TFIIH (60, 61), transcription from these URR-driven G-less templates was inhibited by BE2, 11E2, 16E2, and 18E2, in a dose-dependent manner (Fig. 2). Repression of the E6 promoter could be observed on both homologous and heterologous URR-driven templates, irrespective of the types of E2 proteins used (compare left panels of Fig. 2, A-C). In contrast, the internal control template pML53, which contains the adenovirus major late core promoter linked to a G-less cassette of ~280 nucleotides, did not respond to increasing amounts of E2 proteins, indicating a URR-specific repression mediated by E2 proteins. We had illustrated in our previous studies (28) that repression of the E6 promoter from HPV-11 URR-containing G-less cassette templates was mediated mainly through E2 binding to the promoter-proximal #3 and #4 E2-BSs, which are immediately adjacent to the TATA box of the E6 promoter (see Fig. 1C).

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Transcriptional repression mediated by high and low risk HPV and BPV-1 E2 proteins. A, transcriptional repression of the HPV-11 E6 promoter mediated by different E2 proteins. In vitro transcription was performed in a two-component system comprising pol II holoenzyme and TBP using p11URR-GLess template, which contains the HPV-11 URR spanning nucleotides 7072-7933/1-70, and the internal control template pML53, in the absence () or presence of increasing amounts (2, 10, 50, and 200 ng) of BE2 (lanes 1-5, respectively), 11E2 (lanes 6-10), 16E2 (lanes 11-15), or 18E2 (lanes 16-20). Protein concentrations of different E2 proteins were normalized by Western blotting with anti-FLAG M2 monoclonal antibody (Sigma). The line graph shows the relative transcription intensity of HPV-11 template with different amounts of E2 protein. Relative intensity is defined as the signal intensity, quantified by PhosphorImager analysis, from p11URR-GLess relative to that from the same DNA template performed in the absence of E2 (i.e. the first lane of each reaction set). B, transcriptional repression of the HPV-16 E6 promoter mediated by different E2 proteins. In vitro transcription was performed as described in A, except that a transcriptional template p16URR-GLess containing the HPV-16 E6 promoter spanning HPV-16 nucleotides 7007-7904/1-72 was used. The line graph shows relative transcriptional intensity for the HPV-16 E6 promoter at increasing concentrations for each E2 protein titration. C, transcriptional repression of the HPV-18 E6 promoter mediated by different E2 proteins. In vitro transcription reactions were performed as described in A, except that a transcriptional template p18URR-GLess containing the HPV-18 E6 promoter spanning HPV-18 nucleotides 6929-7857/1-81 was used. The line graph shows relative transcriptional intensity for each HPV-18 E6 promoter signal derived from the titration of each E2 protein.

These comparative studies also revealed several interesting findings. First, 16E2 is apparently a better repressor compared with 18E2, 11E2, and BE2, because 10 ng of 16E2 almost completely inhibited HPV transcription, whereas 10 ng of the other E2 proteins only led to less than 50% of inhibition (compare Fig. 2, A-C, lanes 3, 8, 13, and 18, and the diagrams shown on the right of each panel). Obviously, ~50 ng (i.e. a 5-fold higher amount) of 18E2, 11E2, and BE2 is required to reach a level of repression similar to that achieved by 10 ng of 16E2 (see the diagrams in Fig. 2, A-C). Again, stronger repression of the E6 promoter by 16E2, relative to the other E2 proteins, was observed on both homologous (Fig. 2B) and heterologous (Fig. 2, A and C) HPV templates. Second, animal papillomavirus E2 protein (BE2) could also inhibit HPV E6 promoters as efficiently as HPV E2 proteins (Fig. 2), consistent with many previous studies using the BPV-1 E2 expression plasmid to study HPV promoter regulation by transfection assays (11, 55, 56). Third, the repression activity of 18E2 is more similar to that exhibited by 11E2 and BE2, but not to its closely related family member (i.e. high risk 16E2). This observation could be best explained by the available C-terminal DNA-binding/dimerization domain structures of 16E2, 18E2, and BE2 (see "Discussion").

Transcriptional Activation Mediated by High and Low Risk HPV and BPV-1 E2 Proteins-- To compare the transactivation activity of E2 proteins encoded by HPV and BPV-1, we employed a highly purified in vitro transcription system reconstituted with only recombinant general transcription factors (TFIIB, TFIIE, and TFIIF), general cofactor PC4, and epitope-tagged multiprotein complexes (TFIID, TFIIH, and pol II). It has been shown previously (17) that 11E2 can activate transcription from p2E2(IR)53 DNA template, which contains two copies of the HPV-11 #2 E2-BS linked to a major late core promoter-driven G-less cassette of ~280 nucleotides, but not from an internal control template containing five copies of Gal4-binding sites connected to a similar major late core promoter construct with a longer G-less cassette (Fig. 3A, bottom drawings). When we compared HPV and BPV-1 E2 proteins directly in this reconstituted transcription system, we found that 16E2 was the strongest activator achieving a maximum level of activation at only 5 ng of protein compared with the other E2 proteins (Fig. 3A, compare lanes 5, 13, 23, and 31, and Fig. 3B). BE2 and 18E2 displayed similar levels of activation (Fig. 3A, lane 7 versus lane 34, 11.6- and 13.7-fold activation, respectively), but BE2 reached maximum activation at a lower dose-dependent amount than 18E2 (Fig. 3A, compare lanes 7 and 34, 50 ng of BE2 versus 150 ng of 18E2). 11E2 attained its maximum level of activation at the same dosage as BE2 (Fig. 3A, lane 15, 50 ng of 11E2), but with only 7.4-fold activation. After the maximum level of activation is achieved for each E2 protein, a general decline in fold activation, representing transcriptional squelching, is observed (Fig. 3B). It is clear from both experiments for activation and repression that 16E2 is the dominant activator and repressor compared with 18E2, 11E2, and BE2 in these comparative studies.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Transcriptional activation mediated by different E2 proteins. A, E2-dependent activation assays. Reconstituted in vitro transcription reactions were performed with purified TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and pol II, in the absence () or presence (+) of PC4 and increasing amounts of different E2 proteins using p2E2(IR)53 and pG5MLT templates, as described under "Experimental Procedures." MLP, major core late promoter. B, line graph displaying fold activation for each E2 protein. Fold activation is defined as the signal intensity, quantified by PhosphorImager, from p2E2(IR)53 relative to that from the same DNA template performed in the absence of E2 and PC4 (i.e. the first lane).

DNA-binding Activities of High and Low Risk HPV and BPV-1 E2 Proteins-- Because E2 is a sequence-specific DNA-binding protein, we speculated that the transcription activity of E2 protein may correlate with its DNA-binding activity, thereby accounting for the functional differences in transactivation and repression between 16E2 and the other E2 proteins. To explore this possibility, we performed EMSA using purified E2 proteins and DNA probes derived from promoter-proximal #3 and #4 E2-BSs of respective HPV E6 promoters and a URR region containing #11 and #12 E2-BSs from BPV-1 (63). As shown in Fig. 4A, two protein·DNA complexes (C1 and C2) were detected with each E2 protein when a DNA probe containing E2-BSs #3 and #4 of HPV-11 was used for EMSA. The C1 complex represents an E2 dimer binding to one E2-BS first appearing at low concentrations of E2, whereas the C2 complex is formed between two E2 dimers binding to both E2-BSs and observed at increasing concentrations of E2 protein. At higher concentrations of 16E2 and 18E2, we also observed additional protein·DNA complexes migrating slower than the C2 complex (Fig. 4A, lanes 30 and 40), presumably generated by oligomerization of E2·DNA complexes. An appearance of a band (indicated by an asterisk) below the C1 complex of BE2 (Fig. 4A, lanes 11-20) is likely formed between minor degradation products of BE2 and the DNA probe. Consistent with BE2 being the largest E2 protein in this assay, BE2·DNA complexes migrated slower than HPV E2·DNA complexes. Similar patterns of E2·DNA complexes were also detected with DNA probes derived from HPV-16 (Fig. 4B) and HPV-18 (Fig. 4C). When BPV-1 probe was used, 16E2 and 18E2 showed migration patterns similar to those observed with HPV probes (Fig. 4D, lanes 21-40). The C1 and C2 complexes formed with BE2 apparently dissociated during electrophoresis, causing two clusters of large smears on the gel (Fig. 4D, lanes 11-20). The affinity of 11E2 for the BPV probe is extremely low because no clear complexes could be detected even with the use of 200 ng of protein (Fig. 4D, lanes 1-10), which accidentally led to the formation of large protein·DNA aggregates unable to enter the gel. It is apparent from these EMSAs that the C1 complex was formed at much lower concentrations of 16E2 and 18E2 than 11E2 and BE2 (compare lanes 21-40 with lanes 1-20 of Fig. 4, A-C), indicating that high risk E2 has higher affinity for E2-BSs than 11E2 and BE2.

View larger version (75K): [in this window] [in a new window]   Fig. 4.   EMSA performed with E2 binding to DNA probes containing two E2-BSs derived from different types of HPVs and BPV-1. A, binding of 11E2, BE2, 16E2, and 18E2 to a wild-type HPV-11 E6 promoter-proximal DNA fragment containing two E2-BSs. EMSA was performed in the absence () or presence of increasing amounts of E2 protein (in ng), as indicated above each lane, with a 32P-labeled HPV-11 E6 promoter proximal-probe spanning HPV-11 nucleotides 7902-7933/1-92. The identities of the shifted complexes, as indicated by C1, C2, Oligomer, and *, are described under "Results." B, binding of 11E2, BE2, 16E2, and 18E2 to a wild-type HPV-16 E6 promoter-proximal DNA fragment containing two E2-BSs. EMSA was performed as described in A, except that an HPV-16 E6 promoter probe spanning HPV-16 nucleotides 7868-7904/1-96 was used. C, binding of 11E2, BE2, 16E2, and 18E2 to a wild-type HPV-18 E6 promoter-proximal DNA fragment containing two E2-BSs. EMSA was performed as described in A, except that an HPV-18 E6 promoter probe spanning HPV-18 nucleotides 7834-7857/1-101 was used. D, binding of 11E2, BE2, 16E2, and 18E2 to a wild-type BPV-1 DNA fragment containing two E2-BSs. EMSA was performed as described in A, except that a BPV-1 DNA probe spanning BPV-1 nucleotides 7874-7945/1-85 was used.

To compare and quantify accurately the binding affinities among various E2 proteins for each DNA probe, we calculated the equilibrium binding constant (K_d) estimated from three or more gel shift assays by measuring the reduction of free probe (Table I). Calculations of the equilibrium binding constant clearly indicate that 16E2 has the highest affinity to both homologous and heterologous HPV E6 promoter-proximal E2-BSs, followed in order by 18E2, BE2, and 11E2 (see Table I, K_d of 16E2:18E2:BE2:11E2 = 0.719:1.597:3.239:5.190 for HPV-11 probe, and similar comparisons for HPV-16 and HPV-18 probes). In the case of BPV-1 DNA-derived probe containing two E2-BSs, homologous BE2 bound with the highest affinity (K_d = 1.079) followed by 16E2 (K_d = 1.567), 18E2 (K_d = 8.422), and 11E2 (K_d = 25.360), consistent with results published previously (11). This indicates that there may be some species specificity between the DNA-binding activity of BPV and HPV E2 proteins, which allows BE2 to bind better to BPV-1 DNA and HPV E2 to bind better to HPV DNA. This phenomenon likely reflects inherent variation between HPV and BPV E2-BS sequences with HPV preferring A/T spacer nucleotides (ACCGN₄CGGT, see Fig. 1C), whereas BPV contains more G/C spacer nucleotides within their respective E2-BSs. Differences among E2 recognition of DNA (see "Discussion") and decreased stability of BE2 protein may also contribute to the variation in results from EMSA performed with HPV DNA compared with BPV-1 DNA (7, 64).

Binding Properties of Individual E2-BSs #3 and #4 in the HPV-11 E6 Promoter-proximal Region-- We and others have demonstrated previously that E2-mediated repression of the E6 promoter via inhibition of preinitiation complex formation functions mainly through E2 binding to the promoter-proximal #4 E2-BS (9, 10, 25, 28). To explore the possibility that high risk HPV E2 protein may bind with higher affinity to the promoter-proximal #4 E2-BS than low risk 11E2 and BE2 to mediate transcriptional repression, we performed EMSA using an HPV-11 E6 promoter-proximal DNA probe with mutated #3 but wild-type #4 E2-BS (Fig. 5A). As expected, only one predominant complex (C1), formed via E2 binding to the #4 E2-BS, was detected with HPV-11(7902-92)3M probe by different E2 proteins (Fig. 5A). Surprisingly, additional E2·DNA complexes (C2 and oligomers) were also observed with #3 E2-BS-mutated DNA probe at higher concentrations of 16E2 and 18E2 (Fig. 5A, lanes 10 and 17-20), indicating that either high risk HPV E2 proteins do have higher DNA-binding activities able to overcome half-site mutations, or the flanking sequences surrounding E2 half-site mutations may contribute to E2·DNA recognition and binding. Comparison of the equilibrium binding constants (K_d, Table II) revealed that 16E2 indeed bound with the highest affinity (K_d = 0.871) to HPV-11(7902-92)3M probe, followed by 18E2 (K_d = 2.163), BE2 (K_d = 5.026), and 11E2 (K_d = 9.577). To test whether E2-BSs #3 and #4 in the HPV-11 E6 promoter-proximal region are equivalent for E2 binding because they have the same consensus sequence, ACCGAAAACGGT (see Fig. 1C), we also performed EMSA with DNA probe containing mutated #4 E2-BS (Fig. 5B). The results were nearly identical to the mutated #3 probe except that higher order HPV-16 E2·DNA complexes (C2 and oligomer) were clearly observed (Fig. 5B, lanes 8-10), again suggesting that flanking sequences surrounding the E2-BS mutations indeed contribute to E2·DNA binding, which is consistent with previous studies (65). Although higher order E2·DNA complexes appeared with high risk E2 proteins when individual E2-BSs were mutated, comparison of binding constants for each E2 protein binding to either HPV-11(7902-92)3M or HPV-11(7902-92)4M probe indicates that the binding properties of each E2 protein to #3 and #4 E2-BSs are very similar (Table II, compare 11E2 K_d 9.577 versus 10.261, 16E2 K_d 0.871 versus 0.674, 18E2 K_d 2.163 versus 1.807, and BE2 K_d 5.026 versus 7.569). As a control, additional EMSA was performed with a DNA probe containing mutations at both #3 and #4 E2-BSs (Fig. 5C). As expected, 11E2 was unable to bind to HPV-11(7902-92)34M probe (Fig. 5C, lanes 1-10). Surprisingly, protein·DNA complexes were detected with BE2, 16E2, and 18E2 (Fig. 5C, lanes 18-20, 26-30, and 35-40), albeit at significantly lower affinities than binding to wild-type E2-BSs. These studies further confirmed that high risk HPV E2 proteins indeed have higher DNA-binding activity than low risk E2 protein even to overcome mutations in the half-E2-BS, consistent with previous studies reporting that the C-terminal domain of 16E2 has 180-fold higher affinity for nonspecific DNA than the C-terminal domain of BE2 (66).

View larger version (57K): [in this window] [in a new window]   Fig. 5.   EMSA performed with E2 binding to DNA probes containing mutated promoter-proximal E2-BSs derived from HPV-11. EMSAs were performed as described in Fig. 4, except that 32P-labeled HPV-11(7902-92)3M (A), HPV-11(7902-92)4M (B), and HPV-11(7902-92)34M (C) probes spanning HPV-11 nucleotides 7902-7933/1-92 with mutated #3, mutated #4, and mutated #3 and #4 E2-BSs were used.

Correlation of Transcriptional Repression Activity with DNA-binding Activity between 18E2 and 11E2-- There is a clear distinction between repression activity of 16E2 and both 18E2 and 11E2 proteins (see Fig. 2). This activity correlated well with the binding affinities among HPV E2 proteins to DNA probes derived from the HPV E6 promoter-proximal region, in that 16E2 had the highest affinity followed by 18E2 and then 11E2 (Tables I and II). If there is indeed a direct correlation between binding affinities and E2 repressor activity, 18E2 should be a better transcriptional repressor than 11E2 because it has a higher affinity for promoter-proximal E2-BSs than 11E2 (Table I and Fig. 4). However, this was not clearly observed (Fig. 2). If the mechanism of transcriptional repression at the E6 promoter is caused by disruption of preinitiation complex formation through promoter-proximal DNA binding (9, 10, 25, 28), then one possibility that may account for this lack of difference is that there were not enough titration points to measure the differences in activity between 18E2 and 11E2 adequately. The other possibility is that promoter-distal E2-BSs may sequester E2 protein away from promoter-proximal E2-BSs.

To address these issues, we performed an in vitro transcription assay using the two-component transcription system with an HPV-11 URR-containing G-less cassette template where E2-BSs #2 and #3 were mutated (28) while carefully titrating both 11E2 and 18E2 (Fig. 6A). At low concentrations of 11E2 and 18E2 (e.g. 1 ng), the activity from the HPV-11 E6 promoter is almost reduced by half with 18E2, whereas 11E2 showed negligible reduction in E6 promoter activity (Fig. 6A, compare lane 15 with lane 5). Plotting relative intensity determined by PhosphorImager analysis revealed that there is a significant difference between the repression activities of 18E2 and 11E2 at low amounts of E2 proteins, which indeed correlates with their relative DNA-binding affinities for E2-BSs (Fig. 6B).

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Transcriptional repression of the HPV-11 E6 promoter mediated by 11E2 and 18E2. A, transcriptional repression of the HPV-11 E6 promoter by different amounts of E2 proteins. In vitro transcription was performed in a two-component system as described in Fig. 2A, except that p7862-70(23M)GLess/I (28), a transcriptional template containing HPV-11 URR spanning nucleotides 7862-7933/1-70 with mutations at #2 and #3 E2-BSs, was used. B, line graph of relative intensity for 11E2 and 18E2 proteins. Relative intensity is defined as the signal intensity, quantified by PhosphorImager, from p7862-70(23M)GLess/I relative to that from the same DNA template performed in the absence of E2 (i.e. the first lane of each reaction set).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this report, we described the first comparative analysis of transcription and DNA-binding activities of the full-length high risk and low risk HPV and BPV-1 E2 proteins in well defined cell-free environments devoid of other cellular proteins whose presence in the cell may complicate the studies of intrinsic activities of E2 proteins. We uncovered a direct correlation of transcriptional activity with the DNA-binding activity in which high risk 16E2 is the strongest transcriptional activator/repressor, compared with 18E2, 11E2, and BE2, and shows the highest affinity for both homologous and heterologous HPV E6 promoter-proximal E2-BSs. Our studies thus provide unequivocal comparison of functional properties of E2 proteins encoded by high risk and low risk HPVs as well as BPV-1, which play an important role in virus-induced pathogenesis in both humans and animals.

High Risk E2 Proteins Display More Transcriptional Activity than Low Risk HPV and BPV-1 E2 Proteins-- The full-length E2 protein can function as a transcriptional repressor to inhibit the expression of virus-encoded gene products mainly through promoter-proximal E2-BSs (25, 27, 28, 67-69). We found that, similar to high versus low risk HPV E6 and E7 oncoproteins, there are also distinct differences in the repression activities between high and low risk HPV E2 proteins. Using an in vitro transcription system reconstituted with human pol II holoenzyme and TBP, we defined a strict order of transcriptional repression activity with 16E2 acting as the strongest repressor, followed by 18E2 and BE2, and lastly 11E2 (Figs. 2 and 6). The potency of transcriptional repression activity exhibited by different E2 proteins strongly correlates with their DNA-binding affinities for E2-BSs (Fig. 4 and Table I). 16E2 bound with the highest affinity for the E6 promoter-proximal DNA fragments derived from HPV-11, HPV-16, and HPV-18 and was followed closely, again, by 18E2, BE2, and finally 11E2. The strong DNA-binding activity observed with 16E2 and 18E2 even allows these high risk E2 proteins to bind to DNA probes containing half-site mutations in both promoter-proximal #3 and #4 E2-BSs (Fig. 5), whose binding could not occur with low risk 11E2 protein.

Although comparison of transactivation activity among some high and low risk HPV E2 proteins has been described in previous studies using either transient transfection or in vitro transcription performed with HeLa nuclear extract (11, 55, 56), the presence of numerous unidentified cellular proteins in their assays often complicates the interpretation. Therefore we have developed a highly purified E2-dependent in vitro transactivation system (17) comprised only of essential recombinant general transcription factors (TFIIB, TFIIE, and TFIIF), recombinant general cofactor PC4, and epitope-tagged multiprotein complexes (TFIID, TFIIH, and pol II). Using this reconstituted cell-free transcription system, we found that 16E2 is the most responsive and strongest transactivator compared with 18E2, BE2, and 11E2 (Fig. 3), suggesting that 16E2 may be more effective at recruiting components of the general transcription machinery to the promoter region. This interesting possibility remains to be investigated further.

Significance for the Order of Transcriptional Activity between High and Low Risk HPV E2 Proteins-- In our studies, we found a strict order of both transcriptional activation and repression activity among various E2 proteins. High risk 16E2 displays the highest activation and repression activity, followed by high risk 18E2 and then low risk 11E2. In a recent epidemiological study, 99.7% of 1,000 cases of invasive cervical cancer were HPV DNA-positive with HPV-16 DNA (53%) being the most prevalent followed by HPV-18 DNA (15%) (2, 3). Previous studies have also shown that high risk HPV E6 and E7 are more active in inducing cellular transformation than low risk E6 and E7 (for review, see Refs. 4 and 5). A common hallmark of cervical cancer is viral integration, which often disrupts the E2 open reading frame, leading to the loss of E6 promoter regulation and thus increased the expression of HPV E6 and E7 oncoproteins. We speculate that HPV-16 developed a more transcriptionally active E2 protein to regulate its highly active E6 and E7 oncoproteins tightly and govern the viral life cycle more stringently than low risk HPVs, which express less potent E6 and E7 proteins. Once regulation of the E6 promoter by high risk E2 proteins is lost because of viral integration, cancer may then develop.

Differences in DNA Target Site Discrimination by Papillomavirus E2 Proteins-- Although previous studies of binding site affinities between HPV and BPV-1 E2 proteins revealed little variation (11, 56), we found a distinct order of E2 binding to both wild-type and mutated HPV E6 promoter-proximal E2BSs among 16E2, 18E2, BE2, and 11E2 (Figs. 4 and 5 and Tables I and II). One possible explanation for the variation between our results and previously published studies might be attributed to the DNA probes from which the equilibrium binding constants were calculated. Although we used a much larger DNA probe containing two E2-BSs derived from the natural HPV URRs, others have used much smaller DNA probes containing only one or two E2-BSs with minimal flanking regions (11, 56). Smaller DNA probes would naturally have more inherent DNA flexibility than larger DNA probes, which would favor BE2 binding to more flexible DNA, whereas HPV E2 has a predisposition to bind to pre-bent DNA (7). Variations in Mg²⁺ salt concentrations between DNA-binding experiments may also play a role in the differences in calculating equilibrium binding constants because there is evidence that Mg²⁺ enhances HPV E2 recognition of specific E2-BSs (70). Furthermore, the four-nucleotide spacers within our E2-BSs also vary from previous DNA probes (11), consistent with previous studies identifying that HPV E2 favors spacer nucleotides that are A/T-rich, specifically AATT, whereas BPV E2 does not display an ability to discriminate between spacer nucleotides (7, 71). This may explain our results in which 16E2 favors binding to DNA probes derived from HPV URR, whereas BE2 binds better to BPV-derived E2-BSs that are less A/T-rich (Fig. 4 and Table I), a conclusion in agreement with previous studies (11).

It should not be surprising that E2 proteins vary in binding affinities, considering that there are distinct differences in the quaternary structures of the DNA-binding domains between 16E2 and 18E2 (7). These differences are critical because they dictate the spatial arrangement of side chains presented to the major grooves for DNA sequence recognition (64). Furthermore, structural and biochemical studies of E2 proteins from BPV-1 and HPV-16 have suggested they use different mechanisms to discriminate their DNA targets (71) even though they are highly homologous proteins. Recent E2·DNA-binding studies have also reported that there are significant differences in the binding affinities between the DNA-binding domains of HPV-16 and BPV-1 (66). In fact, BE2 shares more structural similarities to 18E2 than 16E2, whereas 16E2 is structurally more similar to HPV-31 E2 protein (7). This may explain why the transcriptional and DNA-binding activity of 18E2 is more equivalent to BE2 than to its closely related human homolog 16E2. Although BE2 and 18E2 are structurally similar, and they both induce the same global DNA deformation upon binding, their mechanisms of deformation are different. BE2 has a positive charge localized near the C terminus of the recognition helix, whereas 18E2 has a positive charge in the center of its DNA interaction surface, and 16E2 is even less charged all along the DNA interaction surface (7). This would lead to differences in E2·DNA contacts, with 16E2 making fewer contacts with the DNA phosphate backbone than either BE2 or 18E2 (7), which would be consistent with increased DNA-binding specificity. Although the DNA-binding domain sequences of high risk 16E2 and 18E2 share 54% identity and 77% homology, they still differ in DNA binding. Thus it is likely that the DNA-binding domain of low risk 11E2 may vary further in structure from both 16E2 and 18E2, thus contributing to variations in DNA-binding affinity among high and low risk HPV E2 proteins. This remains to be elucidated once the structure of 11E2 becomes available.

	ACKNOWLEDGEMENTS

We thank L. T. Chow for genomic HPV-18 DNA, R. Kovelman for pCMV-16E2 and pCMV-18E2 plasmids, P. M. Howley for genomic HPV-16 DNA and pdBPV-1 (142-6) plasmid, and K. Chiu along with J. Watson for assistance in plasmid constructions. We are also grateful to Mary C. Thomas for insightful discussion and critical reading of the manuscript.

	FOOTNOTES

^* This work was supported by Grants CA81017 and GM59643 from the National Institutes of Health and Grant RPG-97-135-04-MBC from the American Cancer Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Mt. Sinai Health Care Foundation scholar. To whom correspondence should be addressed: Dept. of Biochemistry, W-409, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4935. Tel.: 216-368-8550; Fax: 216-368-3419; E-mail: c-chiang@biochemistry.cwru.edu.

Published, JBC Papers in Press, September 17, 2002, DOI 10.1074/jbc.M206829200

	ABBREVIATIONS

The abbreviations used are: HPV(s), human papillomavirus(es); BE2, BPV-1 E2 protein; BPV-1, bovine papillomavirus type 1; CREB, cAMP-response element-binding protein; CMV, cytomegalovirus; E2-BS(s), E2-binding site(s); EMSA, electrophoretic mobility shift assay; HPV-11, HPV type 11; HPV-16, HPV type 16; HPV-18, HPV type 18; 11E2, HPV-11 E2 protein; 16E2, HPV-16 E2 protein; 18E2, HPV-18 E2 protein; PC4, positive cofactor 4; pol, polymerase; TBP, TATA-binding protein; TF, transcription factor; URR, upstream regulatory region.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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