Identification of a Novel Transcription Factor Binding Element Involved in the Regulation by Differentiation of the Human Telomerase (hTERT) Promoter

Abstract

Three different cell differentiation experimental model systems (human embryonic stem cells, mouse F9 cells, and human HL-60 promyelocytic cells) were used to determine the relationship between the reduction in telomerase activity after differentiation and the regulation of the promoter for the hTERT gene. Promoter constructs of three different lengths were subcloned into the PGL3-basic luciferase reporter vector. In all three experimental systems, all three promoter constructs drove high levels of reporter activity in the nondifferentiated state, with a marked and time-dependent reduction after the induction of differentiation. In all cases, the smallest core promoter construct (283 nt upstream of the ATG) gave the highest activity. Electrophoretic mobility shift assays revealed transcription factor binding to two E-box domains within the core promoter. There was also a marked time-dependent reduction in this binding with differentiation. In addition, a distinct and novel element was identified within the core promoter, which also underwent time-dependent reduction in transcription factor binding with differentiation. Sitedirected mutagenesis of this novel element revealed a correlation between transcription factor binding and promoter activity. Taken together, the results indicate that regulation of overall telomerase activity with differentiation is mediated at least in part at the level of the TERT promoter and provides new information regarding details of the regulatory interactions that are involved in this process.

TAK1 represses transcription of the human telomerase reverse transcriptase gene

“…Such transcription factors, for example Mad1 and MT-box-binding factor, have been reported to function as repressors of the hTERT promoter that act when cancer cell differentiation is induced by antagonizing the transcriptional activation ability of c-Myc (Tzukerman et al, 2000;Xu et al, 2001;Horikawa et al, 2002). Although our results showed that TAK1 represses the hTERT promoter activity in an E-box-independent manner, we cannot exclude the possibility that TAK1 represses the hTERT promoter even in an E-box-dependent manner.…”

The human telomerase gene: complete genomic sequence and analysis of tandem repeat polymorphisms in intronic regions

“…These studies show that the human and mouse TERT genes are regulated by inducible transcription factors including c-Myc, Sp1 and NFkB (Horikawa et al, 1999;Wick et al, 1999;Wu et al, 1999;Kyo et al, 2000;Yin et al, 2000;Oh et al, 2000;Tzukerman et al, 2000;Poole et al, 2001). However, these studies were not carried out with a complete hTERT gene including all putative regulatory elements and introns.…”

An experimental platform for studying growth and invasiveness of tumor cells within teratomas derived from human embryonic stem cells

“…The human undifferentiated embryonic stem cell clone H9.1 (15) was kindly provided by J. Itskovitz-Eldor (Technion and Rambam Medical Center, Haifa, Israel), and cells were grown on a mitomycin C-treated mouse embryonic fibroblast feeder layer as described (14). The HEY cell line that was initiated from a disaggregated xenograft ovarian tumor (16,17) Histological Analysis.…”

“…When implanted into immunocompromised mice, human embryonic stem (hES) cells developed teratomas containing complex structures, comprising differentiated cell types representing derivatives of all three major embryonic lineages (11)(12)(13)(14). Therefore, we sought to determine whether human cancer cells would grow within such teratomas and display tumorigenic properties that specifically relate to the surrounding human cellular microenvironment, such as invasiveness and recruitment of blood vessels.…”