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.