The Negative Feedback Regulation Of E2F1on C-Myc-induced HTERT Transcription During Tumorigenesis

BackgroundThe crosstalk between c-Myc and E2F1signaling pathway is thought to play aprominent role in cell fate decisions. Studies have determined that the transcriptionalexpression of human telomerase reverse transcriptase (hTERT), can be activated by c-Myc,while its expression can be inhibited by E2F1. Because E2F1is also a direct transcriptionaltarget of c-Myc, we hypothesized that in normal cells there is a feedback regulation, inwhich E2F1negatively regulates c-Myc-induced hTERT transcription, increases the risk oftumorigenesis to some extent.MethodsIn this study, we chose to use human embryonic fibroblast (HEF) cells as the modelsystem, and performed experiments including gene transfection, CHIP and CHIP-re-CHIPetc. to determine whether E2F1can act as a negative feedback regulator of hTERTtranscription in response to c-Myc activation.Results1) The effect of c-Myc activation on the expression of hTERT and E2F1anddownstream signal pathwaysThe increase in c-Myc activity by rAd-c-Myc transfection could enhance expressionsof both E2F1and hTERT in mRNA and protein level in HEF cells. However, intriguingly,no dose-dependent effect on E2F1expression was observed. In severe c-Myc activation,significant E2F1upregulation could be induced, whereas in mild and moderate c-Mycactivation, no significant E2F1upregulation was found. As to hTERT, the increase inexpression level was observed to be a dose-dependent manner but was restricted to alimited amount even when c-Myc activity was greatly increased. We further tested thedownstream signal pathways. As indicated, in the presence of low/moderate c-Mycactivation, the activity of telomerase was increased gradually, while the apoptosis rate changed little. In the presence of high levels of c-Myc activation, the expression of E2F1protein was increased greatly, which subsequently leads to an increased apoptosis rate inHEF cells. Then, we use E2F1ASON to control E2F1expression, in the presence oflow/moderate c-Myc activation, inhibition of E2F1expression could result in an increase ofhTERT expression, while in presence of high c-Myc activation, inhibition of E2F1expression could restrict cells into apoptosis process and further increase the activity oftelomerase. These results indicated that E2F1exhibits a negative feedback regulation onc-Myc-induced hTERT expression.2) E2F1is a transcriptional repressor for hTERT gene expression induced by c-MycactivationWe performed luciferase reporter assay to examine whether E2F1regulates hTERTpromoter activity in HEF cells and found that overexpression of E2F1significantly reducedthe hTERT promoter activity. To detect direct binding of E2F1to the hTERT promoter, wenext performed chromatin immunoprecipitation (CHIP) assay using anti-E2F1antibody,and the results determined that E2F1could directly bind to the hTERT promoter region.Furthermore, to demonstrate that E2F1controls c-Myc-induced hTERT gene expression, weperformed CHIP-re-CHIP assay by using anti-E2F1and anti-c-Myc antibodies, and theresults indicated that E2F1and c-Myc simultaneously associated on the hTERT promoterregion. Finally, when E2F1expression was inhibited by specific antisense oligonucleotides,the qRT-PCR expression level of immunoprecipitated hTERT promoter region wasobserved to have a measurable reduction. These above results indicated that E2F1couldinhibit c-Myc-induced hTERT expression through binding to the region of hTERT promoter,and will not result in tumorigenesis.ConclusionIn normal cells, E2F1exhibits a key negative feedback regulation on c-Myc-inducedhTERT expression, which is critical to control the transmission of c-Myc-mediatedoncogenic signals.