Telomerase activation by human papillomavirus type 16 E6 protein: induction of human telomerase reverse transcriptase expression through Myc and GC-rich Sp1 binding sites

Abstract

High-risk human papillomaviruses (HPVs) immortalize keratinocytes by disrupting the retinoblastoma protein (Rb)/p16 pathway and activating telomerase. The E7 oncoprotein targets Rb, while the E6 oncoprotein induces telomerase activity in human keratinocytes. This study has examined the mechanism by which E6 activates telomerase. Expression of human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, was found to be increased in keratinocytes stably expressing HPV type 16 E6, suggesting that E6 acts to increase hTERT transcription. hTERT expression and telomerase activity were activated to significantly higher levels in cells expressing both E6 and E7 than in cells expressing E6 alone. This indicates that E7 may augment E6-mediated activation of hTERT transcription. In transient-transfection assays using hTERT reporters, the induction of hTERT expression by E6 was found to be mediated by a 258-bp fragment of the hTERT promoter, proximal to the ATG initiation codon. Previous studies have demonstrated that overexpression of Myc can activate hTERT expression, suggesting that Myc may be a mediator of E6-mediated hTERT induction. However, in cells stably expressing E6, no strict correlation between the level of Myc and the activation of hTERT was found. Consistent with this observation, mutation of the two Myc binding sites in the hTERT promoter only modestly reduced responsiveness to E6 in transient reporter assays. This indicates that activation of Myc-dependent transcription is not essential for E6-mediated upregulation of hTERT expression. The hTERT promoter also contains five GC-rich elements that can bind Sp1. Mutation of these sites within the 258-bp fragment partially reduced hTERT induction by E6. However, when mutations in the Sp1 sites were combined with the mutated Myc binding sites, all activation by E6 was lost. This indicates that it is the combinatorial binding of factors to Myc and Sp1 cis elements that is responsible for hTERT induction by E6.

FIG. 1

Analysis of telomerase activity, hTERT mRNA levels, and Myc protein levels in HFKs stably expressing HPV-16 E6, E7, E6/E7, or Myc. Panels represent four independently infected sets of HFKs. Telomerase activity in whole-cell extracts was measured with the TRAP assay. RT-PCR was performed on total RNA to determine hTERT and GAPDH mRNA levels. Myc protein levels in whole-cell extracts were determined via Western analysis.

FIG. 2

Activation of hTERT promoter-reporter plasmids by E6. (A) Structure of TL 800 and TL DM. Each reporter contains approximately 800 bp of the hTERT promoter fused to the firefly luciferase gene. TL DM contains a point mutation (CACGTG→CACCTG) in each of the two Myc binding sites, located at −242 and −34 relative to the ATG initiation codon. (B) C33A cells were transfected with TL 800 and either pSG5-Myc (Myc), pSG5-16E6 (E6), pSG5-16E7 (E7), or both pSG5-16E6 and pSG5-16E7 (E6/E7) (P < 0.05 for Myc, E6, E7, and E6/E7 when compared to control). (C) C33A cells were transfected with TL 800 or TL DM and either pSG5-Myc or pSG5-16E6 (P < 0.05 for Myc and E6 with TL 800 when compared to control; P < 0.05 for E6 with TL DM when compared to control). (D) HFKs were transfected with TL 800 or TL DM and either pSG5-Myc, pSG5-16E6, or both pSG5-16E6 and pSG5-16E7 (P < 0.05 for Myc, E6, and E6/E7 with TL 800 when compared to control; P < 0.05 for E6-E7 with TL DM when compared to control). Total DNA was equalized with pSG5 in each transfection. Cells were harvested 48 h posttransfection, and luciferase activity was normalized to total cellular protein concentration. Results are the means ± standard deviations of data from at least three experiments.

FIG. 3

Specificity of hTERT promoter activation by E6. (A) HFKs were transfected with TL 800 and either pSG5 (control), pSG5-16E6 (E6), pSG5-16E6Δ9–13 (E6 Δ9–13), or pSG5-11E6 (11-E6) (P < 0.05 for E6, E6 Δ9–13, and 11 E6 when compared to control; P < 0.05 for E6 Δ9–13 and 11 E6 when compared to E6). (B) HFKs were transfected with PXP₂ and either pSG5 (control), pSG5-Myc (Myc), or pSG5-16E6. (C) HFKs were transfected with Fos-luc and either pSG5 or pSG5-16E6. Total DNA was equalized with pSG5 in each transfection. Cells were harvested 48 h posttransfection, and luciferase activity was normalized to total cellular protein concentration. Results are the means ± standard deviations of data from at least three experiments.

FIG. 4

Deletion analysis of hTERT promoter activation by E6. (A) Schematic of hTERT promoter deletion mutants. Deletions were created by recombinant PCR. (B) HFKs were transfected with hTERT promoter-reporter plasmids and either pSG5 (control), pSG5-Myc (Myc), or pSG5-16E6 (E6). Cells were harvested 48 h posttransfection, and luciferase activity was normalized to total cellular protein concentration. Results are the means ± standard deviations of data from at least three experiments. P < 0.05 for Myc with TL 800, TL 684, TL 624, TL 439, and TL 275 when compared to control; P < 0.05 for E6 with each reporter when compared to control.

FIG. 5

Mutational analysis of individual Sp1 binding sites in the hTERT promoter. (A) Schematic of hTERT promoter region containing Sp1 binding sites. (B) HFKs were transfected with hTERT promoter-reporters and either pSG5 (control) or pSG5-16E6 (E6). Cells were harvested 48 h posttransfection, and luciferase activity was normalized to total cellular protein concentration. Results are means ± standard deviations of data from five experiments. P < 0.05 for E6 with each reporter when compared to control.

FIG. 6

Effects of mutations in Sp1 binding sites and Myc binding sites. (A) C33A cells were transfected with hTERT promoter-reporter plasmids and either pSG5 (control) or pSG5-16E6 (E6) (P < 0.05 for E6 with WT 181 and Sp1 MT1–5 when compared to control). (B) HFKs were transfected with hTERT promoter-reporter plasmids and either pSG5 or pSG5-16E6 and pSG5-16E7 (E6/E7) (P < 0.05 for E6-E7 with WT 181 and Sp1 MT1–5 when compared to control). Cells were harvested 48 h posttransfection, and luciferase activity was normalized to total cellular protein concentration. Results are means ± standard deviations of data from three experiments.