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. 2003 Sep;77(18):9852-61.
doi: 10.1128/jvi.77.18.9852-9861.2003.

Human papillomavirus type 16 E6 activates TERT gene transcription through induction of c-Myc and release of USF-mediated repression

H R McMurray  1 , D J McCance
Affiliations

Human papillomavirus type 16 E6 activates TERT gene transcription through induction of c-Myc and release of USF-mediated repression

H R McMurray et al. J Virol. 2003 Sep.

Abstract

Human papillomavirus type 16 (HPV-16), a DNA tumor virus, has a causal role in cervical cancer, and the viral oncoproteins E6 and E7 contribute to oncogenesis in multiple ways. E6 increases telomerase activity in keratinocytes through increased transcription of the telomerase catalytic subunit gene (TERT), but the factors involved in this have been elusive. We have found that mutation of the proximal E box in the TERT promoter has an activating effect in luciferase assays. This suggested that a repressive complex might be present at this site. HPV-16 E6 activated the TERT promoter predominantly through the proximal E box, and thus, might be acting on this repressive complex. This site is specific for the Myc/Mad/Max transcription factors as well as USF1 and USF2. Addition of exogenous USF1 or USF2 repressed activation of the TERT promoter by E6, dependent on the proximal E box. Using siRNA against USF1 or USF2 allowed for greater activation of the TERT promoter by E6. Conversely, loss of c-Myc function, through a dominant-negative Myc molecule, reduced activation by E6. Chromatin immunoprecipitations showed that in the presence of E6, there was a reduction in binding of USF1 and USF2 at the TERT promoter proximal E box, and a concomitant increase in c-Myc bound to this site. This shows that a repressive complex containing USF1 and USF2 is present in normal cells with little or no telomerase activity. In E6 keratinocytes, this repressive complex is replaced by c-Myc, which corresponds to higher levels of TERT transcription and consequently, telomerase activity.

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Figures

FIG. 1.
FIG. 1.
E6 activates the TERT promoter in keratinocytes. (A) Schematic of promoter constructs used: 800 WT, core TERT promoter region from −799 to + 1; 800 MT, mutation of the MT box, located at positions −32 to −23; 800 PE, mutation of the proximal Ebox located at positions −34 to −29; 800 DE, mutation of the distal E box located at positions −241 to −236; 800 PE/DE, mutation of both E boxes. Sequences of each binding site are shown, and mutations are indicated in larger, bold text for each construct. (B and C) Luciferase assays were carried out using mutants shown in panel A. One hundred nanograms of each promoter construct was cotransfected with 1.5 μg of vector (B) or E6 (C). Data shown are from one experiment, representative of three performed. Standard deviations (error bars) and P values are derived from three replicates within one experiment. Shown are the basal activity from each promoter, normalized to wild type (B), and activation by E6 relative to the basal activity of a given promoter construct (C).
FIG. 2.
FIG. 2.
Exogenous USF1 or USF2 can repress E6-mediated activation of TERT promoter through the proximal E box. (A and B) One hundred nanograms of the wild-type TERT 800 promoter construct (A) or the PE mutant promoter (B) and 1 μg of vector or E6 were cotransfected into HFK with 333 or 500 ng of USF1 or USF2 or 333 ng of each together. Data shown are from one experiment, representative of three performed. Standard deviations (error bars) and P values are derived from three replicates within one experiment.
FIG. 3.
FIG. 3.
Reduction in USF1 or USF2 expression potentiates E6 activation of the TERT promoter. (A) Expression of siRNA USF1 or siRNA USF2 reduces levels of these proteins in keratinocytes. Western blots were done of keratinocytes cotransfected with an enhanced GFP expression construct and either the siRNA USF1 or siRNA USF2 molecule and were sorted to isolate transfected cells from nontransfected cells. Numbers below blots represent relative protein amounts. Max is shown as a loading control. (B to E) Reduction in USF1 or USF2 levels allows for greater TERT promoter activation by E6, dependent on the proximal E box. Luciferase assays were performed on the wild-type TERT promoter (B and D) or the PE mutant promoter (C and E) with pSG5 empty vector or E6, in the presence of a control siRNA expression vector, or the anti-USF1 (B and C) or anti-USF2 (D and E) siRNA molecules. Data shown are from one experiment, representative of three performed. Standard deviations (error bars) and P values are derived from three replicates within one experiment.
FIG. 4.
FIG. 4.
Myc levels are increased in E6 keratinocytes. Extracts of E6 keratinocytes, or matched vector controls, were used for a TRAP assay and Western blots. (A) Whole-cell lysates were used for TRAP assays and Western blots for c-Myc, USF1 and USF2. Numbers below c-Myc blots indicate relative amounts of protein, as quantitated by the ChemiImager 5500. Max is shown as a loading control. RT-PCR of E6 is shown to indicate that E6 was expressed as expected. Spliced forms of E6 are readily detected by these primers and are indicated in the figure. GAPDH is shown as a control for the RT-PCRs. (B) Nuclear extracts were used for Western blots of c-Myc, USF1, USF2, and Max (as a loading control). Numbers below Myc blots indicate relative amounts of protein, quantitated as above.
FIG. 5.
FIG. 5.
Loss of c-Myc function inhibits E6 activation of TERT promoter. (A) Luciferase assays were performed with 100 ng of the wild-type TERT promoter cotransfected with 1.3 μg of vector or E6 and 12.5, 50, or 100 ng of a dominant-negative Myc construct in HFK. Data shown are from one experiment, representative of three performed. Standard deviations (error bars) and P values are derived from three replicates within one experiment. (B) As a control, an irrelevant promoter, the Gal4-luciferase, was used to show that normal cellular function was not disrupted by the dominant-negative Myc molecule. One hundred nanograms of Gal4-luc was cotransfected with 100 ng of vector or p300 (1-743)-Gal4 fusion protein and 100 ng of DN Myc.
FIG. 6.
FIG. 6.
Chromatin immunoprecipitation of the TERT promoter. Chromatin was prepared from Babe or E6 keratinocytes and immunoprecipitated with antibodies against USF1, USF2, c-Myc, or GST as a control. Coprecipitated DNA was used for PCRs with primers flanking the proximal E box of the TERT promoter (upper panel), or the INV promoter, as a control (lower panel). Data from one experiment, of four performed, are shown.
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