HPV16-E6 associated hTERT promoter acetylation is E6AP dependent, increased in later passage cells and enhanced by loss of p300

Abstract

The E6 oncoprotein from high-risk HPV types activates human telomerase reverse transcriptase (hTERT) transcription in human keratinocytes. Studies on how E6 regulates hTERT have implicated E-box or X-box elements in the hTERT promoter (Veldman et al., Proc Natl Acad Sci USA 2003;100:8211-14; Oh et al., J Virol 2001;75:5559-66; Gewin et al., Genes Dev 2004;18:2269-82), but the mechanism of activation by E6 is still controversial and not well defined. Here, we demonstrate that induction of both hTERT expression and telomerase activity by HPV-16 E6 in early passage keratinocytes is associated with acetylation of histone H3 at the hTERT promoter, is dependent on the E6 associated protein (E6AP) and is not exclusively reliant on E-box or X-box elements. Further increases in histone acetylation of the hTERT promoter and hTERT transcriptional activity in E6 expressing cells that had been passaged extensively in culture were found to occur only with the endogenous promoter and not with an exogenously introduced hTERT promoter construct. Telomerase activity at both early and late passages, however, was dependent on E6AP expression, implying a continued reliance on E6 function for telomerase activity. Our results demonstrate that E6 induces hTERT promoter acetylation, but that further increases in telomerase activity and histone acetylation in later passage E6 expressing cells are independent of E6 activation of the core hTERT promoter. We also provide evidence that the transcription factor p300 is a potential repressor of telomerase activation and histone acetylation in the context of E6 expression. These studies give insight into how immortalization by HPV results in upregulation of hTERT and furthers our understanding of how telomerase is activated during the process of malignant transformation.

Figure 1

Histone H3 acetylation and activation of the endogenous hTERT promoter by E6 are enhanced at late passage. (a) RT-PCR analysis of hTERT transcript levels indicates induction of hTERT transcript accumulation by E6 and an increased accumulation of hTERT transcript in late passage E6/E7 immortalized HFKs. Actin RT-PCR serves as a loading and normalization control. (b) TRAP analysis shows induction of telomerase activity with E6 expression. Like hTERT transcript accumulation, telomerase activity is enhanced approximately 2-fold in late passage E6/E7 immortalized cells when compared to early passage E6/E7 expressing cells. Positive control extract (pos) is provided by the manufacturer of the TRAP kit (Roche). The value for telomerase activity in HFKs in our assay was set to “1” and the values for all other cell types represent fold changes over HFK. Buffer controls were set as a 0 baseline and subtracted from all other values. (c) ChIP assays specific for acetyl H3-associated hTERT promoter were conducted on HFKs stably expressing HPV oncoproteins. Values for acetylation are expressed as percent acetyl-H3 immuoprecipitation signal relative to the preimmunoprecipitation input signal. ChIP assay using primers specific to the endogenous hTERT promoter shows no acetylation in E7 expressing HFKs, while E6/E7 expressing HFKs produce a signal strength of 17% of the input. Histone H3 acetylation is enhanced to 37% in late passage E6/E7 expressing HFKs. This represents a 2.2-fold increase over early passage cells. Background (bkgd) samples were not incubated with an immunoprecipitating antibody and actin antibody was used as an additional negative immunoprecipitation control. The data shown are representative of several experiments.

Figure 2

E6 expression results in acetylation and activation of an exogenous hTERT promoter equally at early and late passage. (a) Luciferase assays for hTERT promoter activity were performed on HFKs stably transduced with LXSN:E7 or LXSN:E6/E7 at early (E6E7E) or late passage (E6E7L). Luciferase activity values were expressed as fold changes relative to that exhibited by E7 cells. Error bars represent 1 standard deviation from the mean. hTERT promoter activity was induced approximately 5-fold over E7 expressing cells in both early and late passage E6/E7 expressing cells. (b) Chip assays were performed in HFKs using primers specific to a transiently introduced hTERT core promoter. While only a 3% signal was obtained for E7 expressing cells, early and late passage E6/E7 expressing cells gave signals of 19 and 24% respectively. The data shown are representative of multiple assays.

Figure 3

E6 retains its ability to activate the hTERT promoter when an E-box mutated, myc-unresponsive construct (TERT-E-luc) or X-box mutated construct are used. (a) E-box and X-box mutants contain transversion mutations at conserved binding sequences in the promoter. (b) Luciferase assays were performed with transiently transfected wild-type TERT-luc or mutant TERT-E-luc constructs in triplicate. Error bars represent 1 standard deviation from the mean. Activation of the hTERT promoter by a c-Myc expression construct is abolished with the E-box mutation. (c) HFKs were transfected with pGL3-basic, TERT-luc, TERT-E-luc or TERT-X luciferase reporter vectors respectively. Activation of the promoter constructs by E6 when compared to non-E6 expressing vector controls is expressed as percentage of full activation of the wild-type promoter by E6. Approximately 65% activation by E6 is retained when the E-box is mutated. Mutation of the X-box element of the hTERT promoter (TERT X-luc) results in no significant decrease in activation by E6. Where not indicated, E6E7 expressing HFKs are at early passage. Experiments were performed in triplicate. Error bars represent 1 standard deviation from the mean.

Figure 4

shRNA expression effectively knocks down transcript and protein levels for E6AP and p300 targets. (a) RT-PCR analysis shows greatly diminished levels of E6AP transcript in E6/E7 expressing HFKs when E6AP shRNA is stably expressed from retroviral vectors. shRNA of a random, nonspecific sequence (neg) is used as a negative control. Likewise, p300 transcript level is diminished by p300 shRNA expression. (b) Immunoblotting demonstrates greatly reduced levels of E6AP protein under expression of the specific E6AP shRNA. Also, since E6-mediated degradation is dependent on E6AP, knockdown of E6AP in E6/E7 expressing cells rescues p53 accumulation. p300 protein levels are also greatly reduced with p300 shRNA expression. Notably, p300 levels are much lower in late passage cells.

Figure 5

Histone H3 acetylation and activation of the hTERT promoter is dependent on E6AP and enhanced by loss of p300. (a) hTERT transcript is detectable in HFKs expressing a nonspecific shRNA (neg, lanes 3 and 7) using RT-PCR. Accumulation of hTERT transcript is decreased with E6AP knock-down (lanes 1 and 4), and increased when p300 shRNA is expressed (lanes 2, 5 and 6). p300-1 and p300-2 represent 2 different shRNA constructs targeting 2 distinct sequences within the p300 transcript. RT-PCR was performed to detect the presence of E6 transcript. Double bands represent multiple splicing products of the E6 transcript. The RT- row represents no reverse transcriptase negative controls. (b) Telomerase activity measured by TRAP assay shows decreased activity with E6AP shRNA expression when compared to that with nonspecific shRNA expression (neg). Telomerase activity is increased approximately 3-fold with p300 knock-down. (c) A representative ChIP analysis. hTERT promoter associated acetyl histone H3 signal is expressed as the percentage of the preimmunoprecipitation input signal for each given experimental condition. (d) Histone H3 acetylation as measured by ChIP analysis is decreased by 2-fold with E6AP knock-down and increased by 2.3-fold with p300 knock-down. Triplicate ChIP analysis data is shown in graph form. Error bars represent 1 standard deviation from the mean.