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Identification of a novel telomerase repressor that interacts with the human papillomavirus type-16 E6/E6-AP complex
Open accessAbstract
The critical immortalizing activity of the human papillomavirus (HPV) type-16 E6 oncoprotein is to induce expression of hTERT, the catalytic and rate-limiting subunit of telomerase. Additionally, E6 binds to a cellular protein called E6-associated protein (E6-AP) to form an E3 ubiquitin ligase that targets p53 for proteasome-dependent degradation. Although telomerase induction and p53 degradation are separable and distinct functions of E6, binding of E6 to E6-AP strongly correlated with the induction of hTERT. Here, we demonstrate using shRNAs to reduce E6-AP expression that E6-AP is required for E6-mediated telomerase induction. A yeast two-hybrid screen to find new targets of the E6/E6-AP E3 ubiquitin ligase complex identified NFX1. Two isoforms of NFX1 were found: NFX1-123, which coactivated with c-Myc at the hTERT promoter, and NFX1-91, which repressed the hTERT promoter. NFX1-91 was highly ubiquitinated and destabilized in epithelial cells expressing E6. Furthermore, knockdown of NFX1-91 by shRNA resulted in derepression of the endogenous hTERT promoter and elevated levels of telomerase activity. We propose that the induction of telomerase by the HPV-16 E6/E6-AP complex involves targeting of NFX1-91, a newly identified repressor of telomerase, for ubiquitination and degradation.
Checked 2026-11-12 · 1 reference not checked yet
Overview
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Written by AI from the paper’s full text.
TL;DR
HPV-16 E6 induces hTERT/telomerase, and this paper shows E6-AP is required for that induction. A yeast two-hybrid screen identified NFX1; its NFX1-91 isoform represses the hTERT promoter and is preferentially bound, ubiquitinated, and destabilized by the E6/E6-AP complex.
Key findings
- E6-AP is required for E6-mediated telomerase induction: shRNA against E6-AP reduced E6-AP RNA to ∼29% of vector control, abrogated E6-mediated p53 degradation, and disrupted E6-mediated hTERT induction and TRAP activity in HFKs.
- NFX1 has two splice isoforms with opposing hTERT promoter activities: NFX1-123 coactivated with c-Myc, whereas NFX1-91 repressed the hTERT promoter, reducing activity to ∼60% of background alone and robustly repressing c-Myc induction.
- NFX1-91 is preferentially targeted by E6/E6-AP: E6 expression reduced NFX1-91 half-life ∼46%, from 2.5 h to 1.3 h; MG-132 restored levels; NFX1-91 was highly ubiquitinated, and E6 coimmunoprecipitated preferentially with NFX1-91 rather than NFX1-123.
- NFX1-91 knockdown derepressed endogenous hTERT and increased telomerase activity; E6-expressing cells with reduced NFX1-91 showed greater than twofold increased hTERT by RT-PCR and TRAP, and reduced E6-AP increased NFX1-91 half-life from 1.2 to 1.7 h.
Methods
- Primary human foreskin keratinocytes (HFKs) and 293T, HeLa, C33A, and U2OS cells were used with retroviral shRNA/expression constructs; a yeast two-hybrid screen used catalytically defective E6-AP C833A plus E6 against fetal brain and HeLa cDNA libraries.
- Functional assays included hTERT luciferase reporter assays (710 and 219 constructs), EMSA with recombinant His-tagged NFX1 fragments, RT-PCR, TRAP telomerase assays, Western blotting, cycloheximide and pulse-chase half-life measurements, immunoprecipitation, in vivo ubiquitination assays, and SAβ-galactosidase staining.
- Analyses compared promoter activity, protein stability, ubiquitination, and telomerase activity across vector, E6, E6 mutant, and shRNA conditions; data are presented as representative experiments or averages with error bars where indicated.
Limitations
- The paper states that NFX1-91 did not overexpress well despite repeated attempts, so the authors could not demonstrate hTERT promoter repression by increased NFX1-91 expression.
- The paper states that the reciprocal coimmunoprecipitation to precipitate AU1-16E6 with NFX1 antibody did not work, and that no significant telomere-length differences were observed despite increased telomerase activity.
- The paper states that whether NFX1-123 coactivation is integral to E6-mediated hTERT induction remains to be tested, and future efforts will determine whether either NFX1 isoform is differentially bound in vivo in E6-expressing cells.
- Evident from the text: several key binding and overexpression results are supported by “data not shown,” and the model relies heavily on transient assays, shRNA knockdown, and primary HFK systems rather than in vivo tumor models.
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Claims the paper makes
48 Own result · 17 Own interpretation · 12 Check pending · 7 No full text of the sources · 3 Own method · 2 Sources not found · 1 Not checked yet
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Here, we demonstrate using shRNAs to reduce E6-AP expression that E6-AP is required for E6-mediated telomerase induction.”
- Also stated in: § Results · boosted
“These experiments demonstrated that E6AP expression was required for hTERT induction by HPV-16 E6.”
boostedstates a cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Also stated in: § Results · boosted
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Additional in vitro E6/ E6-AP binding studies (data not shown), as well as published data (Table 1), revealed a strong correlation between E6 binding to E6-AP and telomerase activation.”
boostedstates an association- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“NFX1-123 strongly coactivated with c-Myc at the hTERT promoter, whereas NFX1-91 repressed the activity of the promoter.”
boostedstates a cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“This gene actually encoded two splice variants, NFX1123 and NFX1-91 , with opposing activities in hTERT reporter assays (Fig. 2).”
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“NFX1-123 strongly coactivated with c-Myc at the hTERT promoter, whereas NFX1-91 repressed the activity of the promoter.”
plainstates a cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- It may state two separate claims as one. 25
- Probably not a substantial claim. 19
- The quote only partly states this claim. 2
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Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
Rated minor: it would not touch a result — a value no result rests on, a p-value that stays on the same side of the significance level, or a conflict that is clerical, resolved elsewhere in the paper, or outside its own findings.
In the paper
§ Plasmids
“Briefly, oligos containing the 26- to 29-nt shRNA sequence were used in PCR reactions”
§ Plasmids
“The n91sh construct was targeted to the 3 ′ UTR region (5 ′ -TGTGGAACCAGCCCAACTGCCCATCAGTCAA3 ′ ).”
Rated minor: it would not touch a result — a value no result rests on, a p-value that stays on the same side of the significance level, or a conflict that is clerical, resolved elsewhere in the paper, or outside its own findings.
In the paper
§ NFX1-91 binds a putative X box in the proximal promoter of hTERT
“HFK/LXSN and HFK/E6 cells were treated with 25 µg/mL cycloheximide for the indicated time points.”
§ Proteasome inhibition and cycloheximide treatment
“For half-life analysis, HFKs were treated with 25 µM cycloheximide (Calbiochem) and harvested in WE16th lysis buffer at the indicated time points.”
Citations
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The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53
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Cited by
Human Papillomavirus E6 and E7: The Cervical Cancer Hallmarks and Targets for Therapy
Pal, A., & Kundu, R. (2020) · Frontiers in Microbiology · doi:10.3389/fmicb.2019.03116 · Cited by 650
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Metadata
- Authors
- Gewin, L., Myers, H., Kiyono, T., & Galloway, D. A.
- Affiliations
- University of Washington (US)
- Venue
- Genes & Development
- Publisher
- Cold Spring Harbor Laboratory
- Published
- 2004-09-15
- Volume
- 18
- Issue
- 18
- Pages
- 2269-2282
- ISSN
- 0890-9369 (ISSN-L)
- 1549-5477
- PMC
- PMC517520
- OpenAlex
- W2127352755
- Cited by
- 277
- References
- 60
- Citations by year
- 2026: 7 · 2025: 11 · 2024: 8 · 2023: 8 · 2022: 10 · 2021: 10 · 2020: 14 · 2019: 12 · 2018: 14 · 2017: 11 · 2016: 13 · 2015: 13 · 2014: 13 · 2013: 11 · 2012: 17
- Type
- posted content
- Language
- en
- Open access
- green
- pmc.ncbi.nlm.nih.gov
- Locations
- Genes & Development (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (publishedVersion)
- Funders
- National Cancer Institute (T32CA009229, 5 T32 CA 09229-23, R01CA064795)
- National Institute of Allergy and Infectious Diseases (T32AI007140)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Telomeres, Telomerase, and Senescence
- Health Sciences › Medicine › Epidemiology › Cervical Cancer and HPV Research
- Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
- Keywords
- HPV16, E6 oncoprotein, UBE3A, E3 ubiquitin ligase, hTERT promoter, ubiquitination, p53 degradation, epithelial cells
- MeSH terms
- Amino Acid Sequence, Binding Sites, Cells, Cultured, DNA-Binding Proteins, Enzyme Activation, Humans, Molecular Sequence Data, Mutation, Oncogene Proteins, Viral, Promoter Regions, Genetic, Repressor Proteins, Tumor Suppressor Protein p53, Genes, myc, Telomerase, Protein Isoforms, Ubiquitin-Protein Ligases, Macromolecular Substances
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