Dose-dependent regulation of the early promoter of human papillomavirus type 18 by the viral E2 protein

Steger, Gertrud; Corbach, S

doi:10.1128/jvi.71.1.50-58.1997

Cited by 162 publications

(68 citation statements)

References 49 publications

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“…Mutation of all three promoter-proximal E2 BS fully relieved E2-mediated repression of P 105 transcription and led to a three-to sixfold activation of transcription. This activation probably depends on the binding of E2 to E2 BS 4 as recently suggested (32). We thus confirmed that E2-mediated repression was dependent only on the integrity of E2 BS 1 in C33 cells while it required two additional binding sites (E2 BS 2 and 3) in HeLa and HaCaT cells.…”

Section: Resultssupporting

confidence: 89%

Different mechanisms contribute to the E2-mediated transcriptional repression of human papillomavirus type 18 viral oncogenes

Demeret

Desaintes

Yaniv

et al. 1997

J Virol

106

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Transcription of the human papillomavirus type 18 (HPV18) E6 and E7 oncogenes is repressed by the viral E2 protein. In C33 cells, we have previously shown that of the four E2 binding sites (E2 BS) present in the HPV18 long control region (LCR), only the binding site adjacent to the TATA box (E2 BS 1) was involved in E2-mediated repression. In the present study, we sought to determine whether this phenomenon was conserved in other cell lines. We first showed that all three E2 BS proximal to the P 105 promoter were required for full repression of its activity in HeLa and HaCaT cells. Repression by E2 at E2 BS 2 occurred through the displacement of Sp1. Second, a truncated E2 product, lacking the N-terminal transactivation domain, repressed transcription more efficiently than the full-length protein. Repression was abolished when the Nterminal domain of E2 was replaced by the activation domain of VP16. The VP16-E2 chimeric protein could activate transcription from an LCR mutated in its TATA box. DNA-protein binding studies showed that E2 associates with its four binding sites in the LCR with similar affinities. However, challenge of such complexes with excess binding sites demonstrated that interaction with E2 BS 4 was the most stable while interaction with E2 BS 1 was the least stable. Furthermore, complexes with the full-length E2 were less stable than those formed with the N-terminally truncated protein.on July 10, 2020 by guest http://jvi.asm.org/ Downloaded from FIG. 7. Dissociation of E2-DNA complexes in the presence of an excess of nonradioactive specific oligonucleotide. Labelled oligonucleotides, at a final concentration of 2 ϫ 10 Ϫ10 M, were incubated with a fixed final concentration of 7 ϫ 10 Ϫ9 M E2DBD or 4 ϫ 10 Ϫ9 M full-length E2. After the reactions reached equilibrium (time point zero), a 1,000-fold excess of the unlabelled oligonucleotide containing E2 BS 4 was added. Aliquots were then taken at various time points and loaded on 6% polyacrylamide running gels. The core sequences of the four E2 BS are indicated under the graph; the palindromes are underlined. The two flanking nucleotides of each sequence, which are involved in the stability of the interaction, are shown in boldface. The half-lives of the complexes (t1/2) determined from these experiments are also given at the bottom. 9348DEMERET ET AL. J. VIROL.

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Section: Resultssupporting

confidence: 89%

Different mechanisms contribute to the E2-mediated transcriptional repression of human papillomavirus type 18 viral oncogenes

Demeret

Desaintes

Yaniv

et al. 1997

J Virol

106

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“…With consequently rising concentrations, E2 also binds to the lowaffinity proximal E2BS3 and E2BS4. This represses transcription from the p97 promoter and thereby maintains the levels of E6 and E7 at a low and autoregulated level as shown in Figure 1A 9,10,12 and as reviewed by von Knebel Doeberitz and Vinokurova 11 (note that the numbering of E2BSs in the study by von Knebel Doeberitz and Vinokurova 11 differs from that currently used).…”

Section: Introductionmentioning

confidence: 92%

“…7 The most important transcriptional regulator of E6 and E7 oncogene expression is the viral E2 protein, which binds to 4 E2-binding sites (E2BSs1, 2, 3, and 4) in the URR. 8 The current model of E2 function in the host cells during the normal viral life cycle implies that E2 binds with high affinity to the E2BS1, resulting in activation of the p97 early promoter even at low E2 concentrations, 9,10 as reviewed by von Knebel Doeberitz and Vinokurova. 11 This promoter activation results in enhanced expression of the early viral proteins, including the oncoproteins E6 and E7 and E2 itself.…”

Section: Introductionmentioning

confidence: 99%

Methylation status of HPV16 E2‐binding sites classifies subtypes of HPV‐associated oropharyngeal cancers

Reuschenbach

Huebbers

Prigge

et al. 2015

Cancer

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BACKGROUND:The human papillomavirus (HPV) E2 protein is a transcriptional repressor of the oncogenes E6/E7 and loss of E2 function is considered a key step in carcinogenesis. Integration of HPV into the host genome may disrupt the E2 gene. Furthermore, methylation of CpG dinucleotides in E2-binding sites (E2BSs) in the HPV upstream regulatory region may interfere with transcriptional repression of E6 and E7 by E2. The authors hypothesized that the CpG methylation status of E2BS identifies subtypes of HPV type 16 (HPV16)-associated oropharyngeal squamous cell cancers (OPSCC) in association with E2 gene integrity and viral integration. METHODS: Methylation of 10 CpG dinucleotides within the upstream regulatory region, encompassing E2BSs 1, 2, 3, and 4, was quantitatively analyzed by bisulfite pyrosequencing in 57 HPV16-associated OPSCC cases. E2 status was analyzed by gene amplification and quantitative real-time reverse transcriptase-polymerase chain reaction. Viral integration was determined by integration-specific polymerase chain reaction methods. RESULTS: Three subgroups with differential methylation at E2BS3 and E2BS 4 were identified: 1) complete methylation (>80%) associated with the presence of integrated HPV genomes with an intact E2 gene; 2) intermediate methylation levels (20%-80%) with predominantly episomal HPV genomes with intact E2; and 3) no methylation (<20%) with a disrupted E2 gene. Patients with high methylation levels tended to have a worse 5-year overall survival compared with patients with intermediate methylation (hazard ratio, 3.23; 95% confidence interval, 1.13-9.24 [P 5.06]). CONCLUSIONS: Methylation of E2BS3 and E2BS4 in OPSCC is associated with E2 integrity and viral physical status. It might explain deregulated viral oncogene expression in the presence of E2. The prognostic significance of E2BS methylation for patients with HPV-associated OPSCC needs to be analyzed further.

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“…If the concentration of the E2 protein rises beyond a certain level, E2 binds also to the low affinity proximal E2BSs (E2BS2, 3 and 4) and represses transcription from the early promoter p97 through displacement of Sp1 and TBP from their respective binding sites, thereby keeping the levels of E6 and E7 under control. [11][12][13] Accordingly, it has been suggested that deregulated expression of E6 and E7 oncogenes and initiation of the transformation process may result from loss of the E2 repressive functions. Viral integration into the host genome usually occurs downstream of the E6 and E7 genes, often in E1 and E2 regions 14,15 and results in a loss of negative-feedback control of oncogene expression.…”

mentioning

confidence: 99%

Differential methylation of E2 binding sites in episomal and integrated HPV 16 genomes in preinvasive and invasive cervical lesions

Chaiwongkot

Vinokurova

Pientong

et al. 2012

Intl Journal of Cancer

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Enhanced expression of the HPV 16 E6-E7 oncogenes may trigger neoplastic transformation of the squamous epithelial cells at the uterine cervix. The HPV E2 protein is a key transcriptional regulator of the E6-E7 genes. It binds to four E2 binding sites (E2BSs 1-4) in the viral upstream regulatory region (URR). Modification of E2 functions, for example, by methylation of E2BSs is hypothesized to trigger enhanced expression of the viral E6-E7 oncogenes. In the majority of HPV-transformed premalignant lesions and about half of cervical carcinomas HPV genomes persist in an extra-chromosomal, episomal state, whereas they are integrated into host cells chromosomes in the remaining lesions. Here we compared the methylation profile of E2BSs 1-4 of the HPV 16 URR in a series of 18 HPV16-positive premalignant lesions and 33 invasive cervical cancers. CpGs within the E2BSs 1, 3, and 4 were higher methylated in all lesions with only episomal HPV16 genomes compared with lesions displaying single integrated copies. Samples with multiple HPV16 integrated copies displayed high methylation levels for all CpGs suggesting that the majority of multiple copies were silenced by extensive methylation. These data support the hypothesis that differential methylation of the E2BSs 1, 3 and 4 is related to the activation of viral oncogene expression in cervical lesions as long as the viral genome remains in the episomal state. Once the virus becomes integrated into host cell chromosomes these methylation patterns may be substantially altered due to complex epigenetic changes of integrated HPV genomes.Persistent infections with high-risk human papillomaviruses (HR-HPVs) are the primary cause of cancer at various sites of the anogenital tract, including the cervix, vulva, vagina, penis and anus, as well as a subset of head and neck cancers. 1 The predominant HR-HPV type in cervical cancers is HPV16, accounting alone for about 50% of all cervical cancers. 2 It is commonly accepted that HPVs infect the basal cells of the squamous epithelium. Oncogenic human papillomaviruses encode two well-characterized oncoproteins, E6 and E7 that are under tight transcriptional control in early permissive, but not yet transformed genital HPV-infections. The enhanced expression of these oncogenes in basal and parabasal squamous epithelial cells of the infected epithelium is the cause of neoplastic transformation of the infected cells. 3,4 The main transforming properties of the E6 and E7 oncoproteins are related to their ability to inactivate the p53 and retinoblastoma (pRB) tumor suppressor proteins, respectively. 4-7 E7 overexpression in HPV-infected cells is accompanied by substantial overexpression of the cyclin-dependent kinase inhibitor p16 INK4a . p16 INK4a overexpression is therefore used as surrogate biomarker for HPV-transformed epithelial cells. 8 Expression of the E6 and E7 genes is regulated by the upstream regulatory region (URR). The HPV 16 URR contains sequences that regulate transcription and replication of the viral genome to which both cell...

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Dose-dependent regulation of the early promoter of human papillomavirus type 18 by the viral E2 protein

Cited by 162 publications

References 49 publications

Different mechanisms contribute to the E2-mediated transcriptional repression of human papillomavirus type 18 viral oncogenes

Different mechanisms contribute to the E2-mediated transcriptional repression of human papillomavirus type 18 viral oncogenes

Methylation status of HPV16 E2‐binding sites classifies subtypes of HPV‐associated oropharyngeal cancers

Differential methylation of E2 binding sites in episomal and integrated HPV 16 genomes in preinvasive and invasive cervical lesions

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