CpG methylation directly inhibits binding of the human papillomavirus type 16 E2 protein to specific DNA sequences

Thain, Alison; Jenkins, Owen; Clarke, AR; Gaston, Kevin

doi:10.1128/jvi.70.10.7233-7235.1996

Cited by 91 publications

(46 citation statements)

References 26 publications

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“…In this regard, methylation of CpG dinucleotides within the E2BSs in the HPV URR has been demonstrated to alter binding affinity of the E2 protein, to activate the p97 promoter, and, consequently, to enhance E6 and E7 transcription in the presence of E2. [23][24][25] This role of methylation is supported by in vitro models demonstrating a hyperactivation of the p97 promoter at the time of E2 binding to the methylated high-affinity E2BS1 24 and parallel disruption of promoter inhibition by impaired binding of E2 to methylated low-affinity E2BS3 and E2BS4 ( Fig. 1B).…”

Section: Introductionmentioning

confidence: 59%

“…1B). 23 We hypothesized that CpG methylation of the activating E2BS1 and repressive E2BS3 and E2BS4 in the . Schematic illustration of the human papillomavirus (HPV) upstream regulatory region with its E2-binding sites (E2BSs) and CpG dinucleotides with regard to (A) the regulation of E6/E7 expression by binding of E2 to E2BSs and (B) the consequence of methylation.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Introductionmentioning

confidence: 59%

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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“…Similarly, within the promoter region of HPV16, we observed increased methylation at E2BS3 as well as a tendency for methylation within the E2BS4 of the xenograft, which could possibly reduce the ability of the E2 to repress transcription of E6 and E7 genes. 50,51 Despite increased methylation at these E2BS sites, the level of E7 mRNA expression was rather modest in the xenograft.…”

Section: Discussionmentioning

confidence: 97%

Establishment and characterization of a human papillomavirus type 16–positive tonsillar carcinoma xenograft in BALB/c nude mice

et al. 2015

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“…40 The data reported in this study suggest that methylation of the proximal E2 binding sites (E2BS3 and 4) in addition to methylation of the E2BS1 might contribute to the transformationassociated activation of the URR in the episomal HPV16 genome most likely due to loss of binding of E2 to the repressive low affinity binding sites E2BS3 and 4. 29,30 If the HPV16 genome becomes integrated and E2 ORF is disrupted or lost, methylation of E2BSs more likely does not bring any selective advantages for the cell clones in the absence of intact E2.…”

Section: Discussionmentioning

confidence: 99%

“…28 Previous reports demonstrated that the capacity of the E2 protein to bind the consensus E2BS sequence in vitro is blocked by methylation of CpG dinucleotides. 29 These data suggested that methylation of E2BSs may interfere with the regulatory functions of E2 upon binding to these sites and potentially could activate transcription of the downstream early genes including E6 and E7. 30 The first evidence that DNA methylation is an important regulatory mechanism in modulating HPV expression came from in vitro studies demonstrating that the transcriptional activity of the HPV18 URR can be altered by DNA methylation.…”

Section: What's New?mentioning

confidence: 93%

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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CpG methylation directly inhibits binding of the human papillomavirus type 16 E2 protein to specific DNA sequences

Cited by 91 publications

References 26 publications

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

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

Establishment and characterization of a human papillomavirus type 16–positive tonsillar carcinoma xenograft in BALB/c nude mice

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

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