Histone modification plays a central role in the regulation of hTERT expression in relation to both cell cycle progression and differentiation.11-13,22,23
The use of HDAC inhibitors has been shown by many to activate transcription of hTERT and telomerase activity.12,13,24,25
Sp1, E2F and E-box binding repressors have all been shown to recruit HDACs to the hTERT promoter, resulting in transcriptional repression.11,22,23,26
However, a role for histone modification in E6-mediated hTERT activation has not been previously implicated. Our data indicates that E6, in conjunction with E6AP, induces acetylation of histone H3 at the hTERT promoter. The level of hTERT promoter acetylation is directly proportional to the level of hTERT transcript accumulation and telomerase activity in HFKs stably expressing E6. This provides a novel demonstration that histone modification is an integral part of the mechanism of hTERT promoter activation by E6.
In early passage cells, E6 modestly activates hTERT transcription and telomerase activity, while in later passage cells, much higher telomerase activity is seen. In agreement with this, we have found that late passage, E6E7 immortalized HFKs have higher hTERT promoter acetylation, hTERT transcript levels and telomerase activity than their early passage predecessors. We find that only the endogenous hTERT promoter is affected in a passage-dependent manner, while acetylation and activity of an introduced hTERT core promoter remains stable between early passage and late passage cells. This suggests that it is alterations of the endogenous promoter, independent of the activity of E6 on the hTERT core promoter represented by the exogenous construct, that result in increased telomerase activity at later passage. The mechanism accounting for additional epigenetic modifications at the endogenous promoter in late passage cells is unknown. It is apparent that although telomerase activity is enhanced in late passage cultured cells, late passage hTERT activity remains dependent on E6 and E6AP, as evidenced by the abrogation of late passage hTERT activity in E6 expressing cells with shRNA targeting E6AP.
Aberrant transcriptional regulation by histone modification enzymes is common in many cancers.25,27
The majority of the research being done regarding these potential targets for cancer treatment has focused on the use of HDAC inhibitors. Although certain genes related to proliferation, differentiation and the development of cancer are beneficially regulated by the inhibition of histone acetylation, resultant increases in histone acetylation may cause undesirable phenotypes in the context of cancer and other pathological conditions. Inhibition of histone deacetylation is likely to activate hTERT transcription and telomerase activity, which could support cellular immortalization and proliferation.
Our data indicates that p300 may have repressive effects on hTERT transcription and telomerase activity in the context of hTERT activation by E6. Although E6AP is needed for hTERT promoter activation, p300 is not apparently degraded by E6, nor is knock-down of p300 able to activate transcription of hTERT in non-E6 expressing cells. Also, p300 knockdown was not sufficient to activate hTERT transcription in cells expressing E7 alone. Therefore, it is unlikely that inactivation of p300 is the primary means of hTERT promoter activation by E6. There has never been a direct implication of p300 in regard to the regulation of hTERT transcription or telomerase activity in normal cells, although it does have a regulatory effect on factors that may play a role in hTERT regulation. p300 modifies transcription factors associated with growth arrest and differentiation28,29
and positively regulates their function. Studies have shown that p300 and the related CREB-binding protein have repressive effects on c-Myc30,31
and c-Jun transcription factors,31
both of which have been implicated as playing a positive role in hTERT transcription. Antisense knockdown of p300 in human breast epithelial cells led to the increased expression of c-Myc and c-Jun. Another possible link between p300 and hTERT repression is the E2F family of transcription factors. Modification of E2F by the HAT activity of p300 is necessary for E2F function and transition from G1 to S phase of the cell cycle.32
E2F has recently been found to be an important repressor of hTERT transcription in relation to both cell cycle and differentiation-dependent hTERT regulation.11,22
E2F binding sites within the hTERT promoter represent an attractive candidate for a role in E6 activation of hTERT transcription. This is especially true given our findings that histone acetylation correlates with hTERT activation by E6 and recent data suggesting that E2F recruitment of HDACs is a central regulating mechanism of hTERT transcription in normal cells.11,22
Our current findings provide insights into the mechanism of hTERT activation by HPV-E6 and the increase in hTERT activity in immortalized cells that may implicate possible targets for cancer therapy and prevention.
Previous studies on E6 activation of hTERT suggest the involvement of E-box or X-box elements of the hTERT promoter. Although controversial, it is agreed that the proximal promoter containing E-box and X-box elements contains a repressive element or elements, and that this region of the promoter is responsible for HPV-E6 activation of hTERT transcription. The downstream E-box was the first site to be proposed as the element responsible for E6 activation of hTERT transcription.15,18
However, E6 has been found to have no significant effect on Myc levels in the cell.15,16,18
On the other hand, 1 group has reported that the E-box element was not essential for telomerase activation by E6.16
Others have suggested that USF family of transcription factors represses hTERT through E-boxes and that this event is involved in hTERT regulation by E6.33
More recently, NFX-91 has been implicated as a repressor of hTERT and it has been suggested as a putative target for degradation by E6/E6AP.18
Our data provides evidence that the ability of E6 to activate the hTERT core promoter is not exclusively dependent on the function of either E-box or X-box elements. The possibility remains that an unidentified repressor acts on the proximal core promoter of hTERT and is involved in activation and histone acetylation induced by E6.