Identification of a novel transcription factor binding element involved inthe regulation by differentiation of the human telomerase (hTERT) promoter

Three different cell differentiation experimental model systems (human embr yonic stem cells, mouse F9 cells, and human HL-60 promyelocytic cells) were used to determine the relationship between the reduction in telomerase act ivity after differentiation and the regulation of the promoter for the hTER T gene. Promoter constructs of three different lengths were subcloned into the PGL3-basic luciferase reporter vector. In all three experimental system s, all three promoter constructs drove high levels of reporter activity in the nondifferentiated state, with a marked and time-dependent reduction aft er the induction of differentiation. In all cases, the smallest core promot er construct (283 nt upstream of the ATG) gave the highest activity. Electr ophoretic mobility shift assays revealed transcription factor binding to tw o E-box domains within the core promoter. There was also a marked time-depe ndent reduction in this binding with differentiation. In addition, a distin ct and novel element was identified within the core promoter, which also un derwent time-dependent reduction in transcription factor binding with diffe rentiation. Site-directed mutagenesis of this novel element revealed a corr elation between transcription factor binding and promoter activity. Taken t ogether, the results indicate that regulation of overall telomerase activit y with differentiation is mediated at least in part at the level of the TER T promoter and provides new information regarding details of the regulatory interactions that are involved in this process.